@article {pmid42647693,
year = {2026},
author = {Luboshitz, A and Rahimi, M and Hendel, A and Orenstein, Y},
title = {EpiCRISPR: Improving CRISPR/Cas9 On- and Off-Target Prediction by Flanking Sequences, Epigenetic Marks, and High-Throughput Datasets.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {23},
number = {5},
pages = {2281-2294},
doi = {10.1109/TCBBIO.2026.3727752},
pmid = {42647693},
issn = {2998-4165},
mesh = {*CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; Prediction Algorithms ; },
abstract = {CRISPR/Cas9 technology has revolutionized gene-editing technologies. However, the endogenous gene-editing efficiency at the target site, unintended off-target sites (OTSs), and the DNA repair outcomes are affected by various factors, among them are the sequence of the site, its flanking sequences, and the epigenetic marks harboring it. Researchers have developed computational methods to predict endogenous efficiency, OTSs, and repair outcomes. However, previous studies have failed to successfully incorporate the factors that affect on- and off-target activity, and repair outcomes, and the methods were trained and evaluated on small datasets. A recent dataset of endogenous on-target efficiencies and repair outcomes of nearly 1,600 measurements in T cells, and a recently preprocessed dataset of 78 OTS experiments in T cells provide a unique opportunity to develop a method to predict endogenous on-target efficiency, OTSs, and repair outcomes. Here, we developed EpiCRISPROn and EpiCRISPROff to improve on-target efficiency and OTSs prediction, respectively, by combining various inputs: the site and its flanking sequences, multiple epigenetic marks, and a high-throughput-based prediction. In EpiCRISPROn and EpiCRISPROff, the additional non-sequence features improved prediction performance from an average Spearman correlation of 0.31 to 0.51 in 5-fold cross-validation and from an average AUPRC of 0.436 to 0.441 on a held-out test set, respectively. Moreover, EpiCRISPROn and EpiCRISPROff were trained on one cell type and successfully generalized to other cell types. In contrast, the inclusion of flanking sequences and epigenetic features did not improve repair-outcome prediction. Furthermore, by interrogating EpiCRISPROn and EpiCRISPROff trained models we shed light on on-target and OTS editing preferences. We expect EpiCRISPROn and EpiCRISPROff to advance the field of gene editing by providing improved cell-type-aware prediction of endogenous CRISPR/Cas9 on- and off-target activity.},
}

*CRISPR-Cas Systems/genetics
*Epigenesis, Genetic/genetics
*Gene Editing/methods
Humans
*Computational Biology/methods
Prediction Algorithms

@article {pmid42671082,
year = {2026},
author = {Zhang, Z and Liao, J and Yu, T and Chen, S and Zeng, L and Chen, G and Shen, Q and Tang, Y and Jiang, M and Chen, J and Wang, L},
title = {Lipopolymer Nanoparticle-Mediated In Vivo CRISPR-Cas9 Editing of Mrg15 Restores Mitochondrial Mitophagy to Alleviate Metabolic-Associated Steatohepatitis.},
journal = {Advanced healthcare materials},
volume = {15},
number = {37},
pages = {e71652},
doi = {10.1002/adhm.71652},
pmid = {42671082},
issn = {2192-2659},
support = {52473328//National Natural Science Foundation of China/ ; 2024B1515040006//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B1212060013//Science and Technology Planning Project of Guangdong Province/ ; 2023HYSPT0703//Guangdong High-Level Youth Talent Project/ ; 1320900066//Sun Yat-sen Clinical Scientist Program/ ; },
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Mitophagy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Mitochondria/metabolism ; Mice, Inbred C57BL ; Male ; Liver/metabolism/pathology ; *Fatty Liver/metabolism/genetics/pathology ; Humans ; },
abstract = {Metabolic-associated steatohepatitis (MASH) remains difficult to treat due to the lack of interventions capable of targeting upstream disease drivers and achieving durable disease modification. The epigenetic regulator Mrg15 has been implicated in mitochondrial dysfunction and metabolic stress in the liver, suggesting its potential relevance to MASH pathogenesis. Here, we develop a liver-targeted lipopolymer nanoparticle (LPNP)-mediated gene editing platform to enable in vivo disruption of Mrg15 by co-delivery of Cas9 mRNA and Mrg15 sgRNA. The screened P64H/Mrg15 system achieved efficient hepatic delivery and genome editing, resulting in reduced Mrg15 expression in hepatocytes. In the MASH mouse model, P64H/Mrg15 treatment was associated with decreased hepatic lipid accumulation, improved liver injury markers, and attenuation of inflammation and fibrosis. Sequence-level analyses confirmed on-target editing in liver tissue, and systemic histopathological evaluation revealed no overt toxicity in major organs under the tested dosing regimen. Transcriptomic profiling revealed coordinated pathway-level associations involving metabolic, inflammatory, and autophagy-related regulation, while protein-level analyses demonstrated alterations in selected autophagy- and mitophagy-related regulators, including TUFM and LC3B-II. Together, these findings identify Mrg15 as a disease-relevant epigenetic regulator in MASH and highlight liver-directed P64H LPNP/CRISPR delivery as a promising non-viral strategy for modulating upstream regulatory pathways in metabolic liver disease.},
}

Animals
*Nanoparticles/chemistry
Mice
*Mitophagy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Mitochondria/metabolism
Mice, Inbred C57BL
Male
Liver/metabolism/pathology
*Fatty Liver/metabolism/genetics/pathology
Humans

@article {pmid42715831,
year = {2027},
author = {Ri, L and Cortez, JV and Bogema, DR and Nevard, R and Day, M and Grupen, CG and McFarlane, GR},
title = {Rapid CRISPR-based bovine embryo sexing to streamline genotype-informed cattle breeding.},
journal = {Theriogenology},
volume = {267},
number = {},
pages = {118163},
doi = {10.1016/j.theriogenology.2026.118163},
pmid = {42715831},
issn = {1879-3231},
mesh = {Animals ; Cattle/genetics/embryology ; *Sex Determination Analysis/veterinary/methods ; Male ; Female ; Genotype ; *Breeding/methods ; Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; Genotyping Techniques/veterinary ; *Embryo, Mammalian ; },
abstract = {Cattle in vitro fertilisation and embryo transfer programmes increasingly rely on embryo-level selection to accelerate genetic gain, but current sexing and genotyping workflows can be costly, slow and logistically demanding. This study developed an efficient, low-resource workflow for bovine embryo sexing that combines whole genome amplification (WGA) with recombinase polymerase amplification-CRISPR-Cas12a (RPA-Cas12a). It also assessed whether the same WGA biopsy products could be used for downstream single nucleotide polymorphism (SNP) microarray genotyping. A one-tube RPA-Cas12a assay targeting the bovine Y-chromosome S4 repeat was developed for fluorescence and lateral flow assay (LFA) readouts. Analytical sensitivity was assessed using serially diluted bovine genomic DNA (gDNA), and breed robustness was tested using male and female gDNA from five major beef breeds and Holstein cattle. The workflow was then applied to WGA products from 22 bovine blastocyst biopsies, with sex calls validated against an established real-time PCR melt curve assay and 100K SNP microarray genotyping. The assay detected male bovine gDNA down to 100 pg using both fluorescence and LFA readouts, with no signal from female gDNA. Male-specific detection was consistent across all breeds tested. All WGA-RPA-Cas12a sex calls from blastocyst biopsies were concordant with real-time PCR and SNP microarray sex calls, and WGA biopsy products produced genome-wide SNP call rates above 85%. This workflow provides a practical approach for rapid bovine embryo sex triage and could reduce unnecessary cryopreservation and genotyping while improving the efficiency of genotype-informed cattle breeding programmes.},
}

Animals
Cattle/genetics/embryology
*Sex Determination Analysis/veterinary/methods
Male
Female
Genotype
*Breeding/methods
Polymorphism, Single Nucleotide
*CRISPR-Cas Systems
Genotyping Techniques/veterinary
*Embryo, Mammalian

@article {pmid42759490,
year = {2026},
author = {Li, Z and Xiong, X and Cao, R and Li, JF},
title = {Precision genetic manipulation: CRISPR toolkits reshape plant biology.},
journal = {Plant physiology},
volume = {202},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag696},
pmid = {42759490},
issn = {1532-2548},
support = {32125004//National Natural Science Foundation of China/ ; 2023B0303000022//Guangdong Major Project of Basic Research/ ; 2019YFA0906202//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Plants/genetics ; *Genome, Plant/genetics ; *Genetic Engineering/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The emergence of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized plant genome engineering over the past decade. This review begins by highlighting 4 seminal early CRISPR studies published in Plant Physiology that laid critical foundations for crop genome editing and tool development. We then summarize major advances in optimizing CRISPR performance, expanding targeting scope, and developing strategies for transgene removal and transient editing. We also discuss a wide array of innovative applications beyond gene knockout, such as gene targeting, chromosome engineering, synthetic apomixis, and gene drive. Furthermore, we introduce key CRISPR-derived tools, including base editors, prime editors, and programmable transcriptional and epigenetic regulators. Finally, we outline remaining challenges to stimulate future exploration of the full potential of CRISPR toolkits.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Plants/genetics
*Genome, Plant/genetics
*Genetic Engineering/methods
Plants, Genetically Modified/genetics

@article {pmid42810238,
year = {2027},
author = {Rahimi, M and Miskel, D and Schreiber, M and Habermann, H and Haake, L and Brenig, B and Besenfelder, U and Havlicek, V and Tesfaye, D and Tholen, E and Rings, F and Held-Hoelker, E and Ghanem, N and Benedetti, C and Hoelker, M},
title = {Zygote origin shapes sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in bovine embryos.},
journal = {Theriogenology},
volume = {267},
number = {},
pages = {118215},
doi = {10.1016/j.theriogenology.2026.118215},
pmid = {42810238},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; *Electroporation/veterinary/methods ; *CRISPR-Cas Systems ; *Zygote/physiology ; *Gene Editing/veterinary/methods ; Female ; Ribonucleoproteins/genetics ; },
abstract = {Despite major progress in generation of edited embryos and offspring via CRISPR/Cas9-RNP electroporation, editing outcomes remain highly variable. This variability may be due to intrinsic differences between in vivo-derived (IVV) and in vitro-derived (IVT) zygotes, which may play a more decisive role than previously anticipated. Therefore, this study aimed to determine how zygote origin shapes the sensitivity to electrical voltage field and editing susceptibility after CRISPR/Cas9-RNP electroporation in the bovine model. The present study systematically compared subsequent developmental competence and editing events in IVV- and IVT-derived bovine zygotes across defined electroporation voltages and pulse durations. Notably, CRISPR/Cas9-RNP electroporation of IVV-derived bovine zygotes of comparable age and collected by tubal embryo flushing (10-18 h post ovulation, hpo) resulted in markedly lower survival rates at 25 and 30 V compared to 20 V. In contrast, higher (p < 0.05) editing frequencies were observed at 25 V and 30 V. Clear differences, however, were observed between the two zygote groups. Following CRISPR/Cas9 RNP electroporation, IVV-derived zygotes yielded a higher proportion of unedited blastocysts (p < 0.05), absence of full edited embryos, a significantly increased frequency of monoallelic edits and reduced rates of mosaicism compared to IVT-derived zygotes. Collectively, the results of the present study clearly underscore a significant lower editing efficiency to pulse sensitivity ratio in IVV-derived zygotes compared to their IVT counterparts. To our knowledge, this is the first study to use bovine IVV-derived zygotes for genome editing using RNP-electroporation to elucidate how zygote origin shapes sensitivity to electrical voltage field and editing efficiency.},
}

Animals
Cattle/embryology
*Electroporation/veterinary/methods
*CRISPR-Cas Systems
*Zygote/physiology
*Gene Editing/veterinary/methods
Female
Ribonucleoproteins/genetics

@article {pmid42825048,
year = {2026},
author = {Ali, B and Hafeez, A and Imin, N},
title = {Beyond the Edit: Integrating CRISPR-Cas Genome Editing Into the Cereal Breeding Pipeline.},
journal = {International journal of genomics},
volume = {2026},
number = {},
pages = {1457684},
pmid = {42825048},
issn = {2314-4378},
abstract = {Cereal breeding must deliver complex trait combinations under a changing climate, and CRISPR-Cas editing is presented as the technology that will accelerate this. Existing reviews document the editing toolkit thoroughly, yet few follow an edited allele through the steps separating an edited plant from a released cultivar. This review addresses that gap by treating genome editing as one module within a cereal breeding pipeline and asking, at each interface, whether integration has been demonstrated or only proposed. Evidence was selected against three criteria, namely, that the work was performed in a cereal, that the outcome was measured rather than projected and that genotype and environment were stated. Applying this standard across wheat, rice, maize and barley yields a more qualified picture than current summaries suggest. Multiplex editing is the dominant strategy for polygenic traits, yet recovery of a complete multilocus genotype is limited by the least efficient guide rather than the average efficiency. Doubled haploid production shortens fixation only where several loci segregate independently, and its induction, identification and doubling steps impose losses that offset part of the saving. Marker-assisted selection acts on backcross progeny and on haploid inducers rather than among near-isogenic edited events, and genomic selection combined with editing rests on livestock simulations whose assumptions cereals do not meet. Transformation efficiency, licensing of regeneration technology and jurisdiction-specific approval remain binding constraints. Every module has been demonstrated in some cereal. The full sequence has not and closing that gap needs integration studies rather than further gains in editing precision.},
}

@article {pmid42825966,
year = {2026},
author = {Wang, S and Chen, Q and Lyu, L and Zhao, ZK},
title = {Metabolic unlocking of lycopene accumulation in the oleaginous yeast Rhodotorula toruloides.},
journal = {Biotechnology letters},
volume = {48},
number = {5},
pages = {},
pmid = {42825966},
issn = {1573-6776},
support = {AA24206050//Guangxi Science and Technology Major Program/ ; 22238010//National Natural Science Foundation of China/ ; },
mesh = {*Lycopene/metabolism ; *Rhodotorula/metabolism/genetics ; *Metabolic Engineering/methods ; Geranylgeranyl-Diphosphate Geranylgeranyltransferase/genetics/metabolism ; CRISPR-Cas Systems ; Carotenoids/metabolism ; Fermentation ; Gene Knockout Techniques ; Intramolecular Lyases ; },
abstract = {Lycopene is a high-value carotenoid with broad applications in the food, pharmaceutical, and cosmetic industries. The oleaginous yeast Rhodotorula toruloides naturally synthesizes mixed carotenoids but fails to accumulate lycopene, primarily due to the bifunctional activity of lycopene cyclase/phytoene synthase Car2, which efficiently converts lycopene into downstream cyclic carotenoids. Here, we engineered R. toruloides for efficient lycopene production using a combinatorial strategy that includes pathway blocking, heterologous enzyme screening, precursor enhancement, and fermentation optimization. CRISPR/Cas9-mediated CAR2 knockout abolished endogenous phytoene synthesizing and lycopene cyclizing activities. Heterologous complementation with exogenous phytoene synthase (CrtB) restored phytoene biosynthesis, and the resultant loss of cyclase activity blocked lycopene cyclization, facilitating robust intracellular lycopene accumulation. Screening three heterologous phytoene synthases identified EgCrtB from Euglena gracilis as the superior enzyme. Co-overexpression of endogenous rate-limiting enzymes involved in the mevalonate pathway further boosted precursor supply. Combined with nitrogen-limited cultivation, which synergistically enhanced lycopene accumulation, the final engineered strain achieved a lycopene content of 0.74 mg/g DCW and a titer of 7.24 mg/L. This work verifies that metabolic pathway modification combined with fermentation regulation can effectively drive lycopene synthesis and accumulation in R. toruloides, offering an early-stage proof-of-concept for further advancing lycopene bioproduction in oleaginous yeast.},
}

*Lycopene/metabolism
*Rhodotorula/metabolism/genetics
*Metabolic Engineering/methods
Geranylgeranyl-Diphosphate Geranylgeranyltransferase/genetics/metabolism
CRISPR-Cas Systems
Carotenoids/metabolism
Fermentation
Gene Knockout Techniques
Intramolecular Lyases

@article {pmid42826192,
year = {2026},
author = {Roach, DJ and Sangruji, BP and Bhat, S and Tesfamariam, S and Ben-Zion, I and Bern, M and Bagnall, J and Shoresh, N and Milien, L and Bhattacharyya, RP},
title = {A rapid, inexpensive diagnostic for bacterial pathogen and resistance detection in resource-limited settings.},
journal = {Science advances},
volume = {12},
number = {40},
pages = {eaeb6630},
pmid = {42826192},
issn = {2375-2548},
support = {K08 AI187322/AI/NIAID NIH HHS/United States ; T32 AI007061/AI/NIAID NIH HHS/United States ; UM1 AI104681/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects/isolation & purification ; Resource-Limited Settings ; Rapid Diagnostic Tests ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods/economics ; },
abstract = {Antimicrobial resistance is a major global health threat, with disproportionate impact in regions with limited diagnostic infrastructure. To address this challenge, we developed BADLOCK (Bacterial and AMR Detection by SHERLOCK), a rapid, low-cost molecular diagnostic platform for direct detection of bacterial pathogens and resistance genes from clinical samples. BADLOCK operates as a one-pot CRISPR-Cas13a reaction capable of detecting nine bacterial species and four major resistance genes directly from positive blood culture. It requires only a heat block and a basic fluorometer for operation. We validated BADLOCK on 194 sequentially collected clinical blood culture specimens growing Gram-negative rods, supplemented with 69 mock samples generated from banked isolates enriched for targeted resistance genes. In all, we conducted 2224 individual reactions, achieving 97.6% accuracy (2171 of 2224) at the reaction level. At the assay level, 89.5% (274 of 306) showed perfect or partial concordance with gold-standard species and resistance gene detection, including 255 assays with perfect concordance and 19 with partial concordance (correct detection of at least one pathogen). This included an evaluation of BADLOCK as a potential culture-free diagnostic for urinary tract infections (UTIs), achieving 98.0% reaction-level accuracy. At the assay level, 95.3% (41 of 43) were partially or perfectly concordant with gold-standard detection of both species and resistance genes. To our knowledge, this represents the first demonstration of the CRISPR-Cas13a diagnostic platform on clinical bloodstream infections to date and supports BADLOCK's potential as a practical and scalable solution for rapid pathogen and resistance gene detection in resource-constrained settings.},
}

Humans
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects/isolation & purification
Resource-Limited Settings
Rapid Diagnostic Tests
CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods/economics

@article {pmid42826193,
year = {2026},
author = {Truong, VH and Miller, DJ and Fatma, S and Sheng, Y and Pindi, C and Ahsan, M and Palermo, G and Kellogg, EH},
title = {Transposon end recognition and pairing by I-F3 CRISPR-associated transposase.},
journal = {Science advances},
volume = {12},
number = {40},
pages = {eaee1374},
pmid = {42826193},
issn = {2375-2548},
support = {F31 GM151863/GM/NIGMS NIH HHS/United States ; R01 GM141329/GM/NIGMS NIH HHS/United States ; R01 GM144566/GM/NIGMS NIH HHS/United States ; R35 GM164142/GM/NIGMS NIH HHS/United States ; },
mesh = {*DNA Transposable Elements/genetics ; *Transposases/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; *CRISPR-Cas Systems ; Molecular Dynamics Simulation ; Models, Molecular ; Protein Binding ; },
abstract = {To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Transposon 7 (Tn7)-like transposons typically contain asymmetric left- and right-end sequences that flank and define DNA cargo. However, how the transposase recognizes these different sequences and assembles them into a catalytically competent state for cut-and-paste transposition remains unknown. Here, we present the cryo-electron microscopy structure of the VchCAST transposase TnsB in complex with transposon DNA ends and host factor IHF, along with biochemistry, molecular dynamics, and in vivo analyses. Our structure reveals the stoichiometry and architecture of the assembly, as well as the DNA distortions required to accommodate transposon end asymmetry. Molecular dynamics suggests that these distortions are required for the coordinated assembly of the complex. Physical association of asymmetric left and right ends results in a previously uncharacterized protein-protein interface that is required for transposition efficiency. Our findings explain how transposases regulate the pairing of transposon end sequences for high-fidelity recognition, reveal a model of transposon-end synapsis, and suggest future avenues to engineer DNA cargo for genome-editing applications.},
}

*DNA Transposable Elements/genetics
*Transposases/chemistry/metabolism/genetics
Cryoelectron Microscopy
*CRISPR-Cas Systems
Molecular Dynamics Simulation
Models, Molecular
Protein Binding

@article {pmid42827080,
year = {2026},
author = {Payne, P and Plevka, P and Bollback, JP and Guet, CC},
title = {Herd immunity underlies homologous recombination in stationary phase bacteria.},
journal = {Molecular biology and evolution},
volume = {43},
number = {10},
pages = {},
pmid = {42827080},
issn = {1537-1719},
support = {648440//H2020 European Research Council/ ; LX22NPO5103//European Commission Programme/ ; //National Institute of Virology and Bacteriology/ ; 8J3AT003//Czech-Austrian mobility/ ; },
mesh = {*Homologous Recombination ; *Escherichia coli/genetics/virology/immunology ; Bacteriophage P1/genetics ; },
abstract = {Homologous recombination is ubiquitous across evolution, generating variation through genetic reshuffling. Although bacteria lack meiotic recombination, homologous recombination has been known to extensively shape their genomes. However, the mechanisms and ecological conditions sustaining frequent recombination in bacteria remain unclear. Using an Escherichia coli-P1 phage model system, we reveal how frequent recombination emerges from CRISPR-based herd immunity to a generalized transducing phage (DNA vector). Herd immunity maintains phage-susceptible donor bacteria, recipient immune bacteria, and transducing phage, which promotes chromosomal gene flow that exceeds spontaneous mutation by up to two orders of magnitude. Notably, recombination begins as bacterial cultures enter stationary phase, when canonical mutation supply through population growth and DNA replication is strongly reduced, and recombination can be reinitiated by brief fresh nutrient pulses. Genetic analysis showed that stationary-phase recombination requires recA, recB, and recG, connecting this process to a defined homologous recombination pathway, and suggests a plausible transcriptional regulatory link to transition to nutrient limitation. Bacterial herd immunity therefore has an unexpected evolutionary consequence: it can promote homologous recombination by turning phages into de facto pollinators. This mechanism can help explain how high rates of homologous recombination may be sustained across bacterial populations and suggests that bacterial immune systems can shape molecular evolution not only by blocking infection but also by regulating genetic exchange.},
}

*Homologous Recombination
*Escherichia coli/genetics/virology/immunology
Bacteriophage P1/genetics

@article {pmid42831426,
year = {2026},
author = {Villegas Warren, R and Omura, SN and Leon Castro, D and Südfeld, C and Luteijn, MJ and Morinaga, H and Inoue, WJ and Shuto, Y and Kuijpers, AM and Meulewaeter, F and De Vleesschauwer, D and Staals, RHJ and Nureki, O and Wu, WY and van der Oost, J},
title = {Size and sequence variation of the Lid of the Cas12a nuclease domain results in a nickase phenotype.},
journal = {Nucleic acids research},
volume = {54},
number = {18},
pages = {},
pmid = {42831426},
issn = {1362-4962},
support = {VI.Vidi.203.074/NWO_/Dutch Research Council/Netherlands ; 24bm1223004h0002/NWO_/Dutch Research Council/Netherlands ; bm1123046/NWO_/Dutch Research Council/Netherlands ; JP21km0908001/NWO_/Dutch Research Council/Netherlands ; ERC-AdG-834279/ERC_/European Research Council/International ; //Supporting Drug Discovery and Life Science Research/ ; JP23ama121002//Basis for Supporting Innovative Drug Discovery and Life Science Research/ ; JP23ama121012//Basis for Supporting Innovative Drug Discovery and Life Science Research/ ; SPI 93-537//NWO Spinoza/ ; },
mesh = {*Endodeoxyribonucleases/genetics/chemistry/metabolism ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism ; Escherichia coli/genetics ; *Deoxyribonuclease I/genetics/metabolism/chemistry ; Protein Domains ; Phenotype ; *Bacterial Proteins/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; Models, Molecular ; },
abstract = {CRISPR-Cas12a nucleases typically induce double-strand breaks (DSBs) to initiate genome editing. However, reliance on DSBs risks chromosomal rearrangements and unintended insertions or deletions at off-target sites. Therefore, engineering nickase variants that cleave only a single DNA strand might enable precise editing platforms. We describe an unbiased in vivo screen in Escherichia coli to identify LbCas12a nickase variants. We targeted the RuvC Lid domain for mutagenesis, hypothesizing that altering this region would disrupt the coordination of strand cleavage. While the screen produced many nickase variants with targeted point substitutions (Lid1), it also serendipitously yielded a variant (Lid2) with an unprecedented Lid domain in terms of size and sequence. Biochemical characterization confirms that Lid2 robustly nicks the non-target strand (NTS) with minor cleavage activity observed for the target strand (TS), accumulating predominantly nicked substrates. Cryo-electron microscopy (cryo-EM) at 2.5 Å resolution reveals that, in Lid2, the native α-helical Lid is replaced, uncoupling the concerted cleavage mechanism. Specifically, the remodeled Lid appears to stabilize the NTS in the active site, whereas it hampers the conformational transitions required for subsequent TS cleavage. This robust nickase-phenotype of this Cas12a variant expands the CRISPR-Cas toolbox and offers a new component for precise genome editing applications.},
}

*Endodeoxyribonucleases/genetics/chemistry/metabolism
*CRISPR-Associated Proteins/genetics/chemistry/metabolism
Escherichia coli/genetics
*Deoxyribonuclease I/genetics/metabolism/chemistry
Protein Domains
Phenotype
*Bacterial Proteins/genetics/chemistry/metabolism
CRISPR-Cas Systems
Models, Molecular

@article {pmid42831658,
year = {2026},
author = {Maheshwari, T and Bhattacharya, S and Singh, D},
title = {Progress in polymeric nanoparticle-based treatment approaches for Wnt β-catenin pathway modulation and adenomatous polyposis coli mutant colorectal cancer.},
journal = {Journal of biomaterials science. Polymer edition},
volume = {},
number = {},
pages = {1-69},
doi = {10.1080/09205063.2026.2736154},
pmid = {42831658},
issn = {1568-5624},
abstract = {About 80-90% of sporadic instances of colorectal cancer (CRC) are caused by mutations in the adenomatous polyposis coli (APC) gene, which is one of the earliest and most common molecular events. The constitutive activation of the Wnt/β-catenin signalling pathway caused by APC dysfunction promotes unchecked cellular proliferation, tumour initiation, progression, and treatment resistance. Systemic toxicity, insufficient tumour selectivity, and acquired drug resistance continue to restrict treatment results for advanced colorectal cancer (CRC) despite advancements in surgery, chemotherapy, targeted treatments, and immunotherapy. Novel treatment approaches that can address the molecular anomalies linked to APC dysfunction are therefore desperately needed. In this context, a number of treatment modalities have been studied, including small interfering RNA (siRNA), microRNA, CRISPR/Cas-based gene-editing systems, chemotherapeutic drugs, and combination therapy. Polymeric nanoparticles (PNPs) are among the strategies that have shown promise as preclinical delivery systems and have the potential to be translated into clinical settings. Poly(lactic-co-glycolic acid) (PLGA), chitosan, and PEG-based polymeric nanoparticles (PNPs) offer biocompatibility, controlled release, enhanced tumour accumulation, and protection of therapeutic cargo. In addition to critically assessing PNP-mediated medication and gene delivery techniques that target APC-associated Wnt/β-catenin signalling, this review explores the mechanistic significance of APC in colorectal carcinogenesis. The promise of PNPs to enhance APC-focused treatment in colorectal cancer is highlighted by emphasising targeted delivery, therapeutic precision, biomarker-guided techniques, translational hurdles, and future clinical prospects.},
}

@article {pmid42833553,
year = {2026},
author = {Ata, A and Topuz Ata, D and Odacı, ZT},
title = {Integrating high-resolution proximity labelling with orthogonal interactome benchmarks: Insights from Kinetoplastid systems.},
journal = {Journal of proteomics},
volume = {332},
number = {},
pages = {105739},
doi = {10.1016/j.jprot.2026.105739},
pmid = {42833553},
issn = {1876-7737},
abstract = {Proximity labelling has progressed from a specialised protein interaction methodology into a pivotal component of modern spatial proteomics. This review presents a comprehensive overview of the technological development of proximity labelling methodologies for mapping proximal protein associations, encompassing first-generation BioID platforms to advanced systems including TurboID, XL-BioID, APEX2 and UltraID. Special emphasis is placed on advances in labelling kinetics, enzyme miniaturisation, conditional activation, and spatial precision. The convergence of proximity labelling with orthogonal tag-free strategies, such as size-exclusion chromatography-mass spectrometry and cross-linking mass spectrometry, is further examined alongside emerging CRISPR-Cas9-based endogenous tagging and artificial intelligence-driven structural prediction frameworks. These complementary strategies are discussed in the context of their potential contribution to reshaping next-generation interactomics. With kinetoplastids as an illustrative model system, reported proximity labelling investigations in Leishmania, Trypanosoma brucei, and Trypanosoma cruzi are systematically consolidated. Critical challenges include parasite-specific biotin metabolism, oxidative stress linked with peroxidase-based labelling, limitations in endogenous tagging, and the extensive dark proteome impeding functional annotation. Next-generation technologies and artificial intelligence-driven interactomics are further explored within kinetoplastid biology, while emphasising their broader potential to combine high-resolution proximity labelling, orthogonal tag-free benchmarking, genome engineering, and artificial intelligence-driven structural analysis across various biological systems, thereby advancing comprehensive interactome mapping and functional proteome annotation. SIGNIFICANCE: Interactomics is a key pillar of systems biology, offering the indispensable functional framework that transforms protein catalogues into dynamic and predictive models of biological systems. To the best of our knowledge, this review represents one of the most comprehensive syntheses integrating all reported proximity labelling implementations in Leishmania, Trypanosoma brucei, and Trypanosoma cruzi up to 2026. Its importance lies in bringing together the development of proximity labelling into a multi-layered methodological framework that now includes robust reference datasets, such as the recently reported large-scale physical interactomes of L. donovani (16,095 interactions) and T. cruzi, generated employing SEC-MS and XL-MS methodologies. Through addressing the "dark proteome," this review positions proximity labelling within a broader multi-modal interactomics framework integrating SEC-MS, XL-MS, and AI-assisted structural modelling, while offering a structured roadmap built around next-generation technologies, including PerTurboID for perturbations, iAPEX for redox challenges, and CRISPR-Cas for physiological tagging. Collectively, these advances may facilitate the development of increasingly predictive and spatially resolved interactome models across complex biological systems.},
}

@article {pmid42433021,
year = {2026},
author = {Moyses-Oliveira, M and Liu, Y and Erdin, S and Gao, D and Bhavsar, R and Mohajeri, K and O'Keefe, K and Boone, PM and Xavier, G and Liao, C and Li, A and Yadav, R and Salani, M and Lucente, D and Currall, B and de Esch, CEF and Tai, DJC and Ruderfer, D and Brennand, KJ and Gusella, JF and Talkowski, ME},
title = {CRISPR-engineered deletion of POGZ alters transcription factor binding at promoters of genes involved in synaptic signaling.},
journal = {HGG advances},
volume = {7},
number = {4},
pages = {100652},
pmid = {42433021},
issn = {2666-2477},
support = {R01 MH123155/MH/NIMH NIH HHS/United States ; R01 MH115957/MH/NIMH NIH HHS/United States ; K08 NS117891/NS/NINDS NIH HHS/United States ; RM1 MH132648/MH/NIMH NIH HHS/United States ; R00 NS118109/NS/NINDS NIH HHS/United States ; U01 HG011755/HG/NHGRI NIH HHS/United States ; R01 HD096326/HD/NICHD NIH HHS/United States ; },
mesh = {Humans ; *Transcription Factors/metabolism/genetics ; *Promoter Regions, Genetic ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Transposases/genetics/metabolism ; *Synapses/metabolism/genetics ; Signal Transduction/genetics ; Neural Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Neurons/metabolism ; Protein Binding ; *Synaptic Transmission/genetics ; Nerve Tissue Proteins ; Basic Helix-Loop-Helix Proteins ; },
abstract = {One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDDs) has been that loss-of-function (LoF) mutations in genes that impact transcriptional regulation confer substantial liability to NDDs. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the mechanisms by which POGZ LoF alters early neuronal development. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and bi-allelic POGZ deletions. In hiPSC-derived neural stem cells (NSCs) and Neurogenin-2-induced neurons (iNs), POGZ LoF altered the expression of genes associated with synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes associated with NDDs, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iNs. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with bi-allelic deletions. These shared molecular consequences suggest key points of convergence that connect gene regulation to neuronal function in the etiology of neurodevelopmental pathologies.},
}

Humans
*Transcription Factors/metabolism/genetics
*Promoter Regions, Genetic
Induced Pluripotent Stem Cells/metabolism/cytology
*Transposases/genetics/metabolism
*Synapses/metabolism/genetics
Signal Transduction/genetics
Neural Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
Neurons/metabolism
Protein Binding
*Synaptic Transmission/genetics
Nerve Tissue Proteins
Basic Helix-Loop-Helix Proteins

@article {pmid42587162,
year = {2026},
author = {Liu, Q and Chen, PA and Urs, E and Zhang, S and Arce, MM and Wang, CH and Yan, J and Nguyen, VQ and Li, Z and Seo, J and Kale, N and Peng, F and Luo, Y and Goudy, L and LaFlam, TN and Zhong, H and Modak, C and Dann, E and Jung, JH and Kirane, A and Warner, AB and Quach, BB and Good, Z and Shy, BR and Shifrut, E and Bapat, SP and Allen, GM and Eyquem, J and Fuh, K and Dodgson, SE and Cyster, JG and Marson, A and Carnevale, J},
title = {In vivo genome-wide CRISPR screens of human T cells in solid tumours.},
journal = {Nature},
volume = {658},
number = {8135},
pages = {496-507},
pmid = {42587162},
issn = {1476-4687},
mesh = {Humans ; Animals ; *T-Lymphocytes/immunology/metabolism/cytology ; Mice ; *Neoplasms/immunology/genetics/pathology/therapy ; *CRISPR-Cas Systems/genetics ; GTP-Binding Protein alpha Subunits, Gs/metabolism/genetics/deficiency ; *Genome, Human/genetics ; Female ; Signal Transduction ; Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; Male ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Receptors, G-Protein-Coupled/metabolism ; Gene Knockout Techniques ; },
abstract = {Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that enhance cellular immunotherapy. Yet, many regulators of T cell performance in solid tumours are not revealed in vitro[1,2]. In vivo screening in tumour-bearing mice is more physiological but has been limited by low intratumoural T cell recovery. Here we developed an in vivo model that efficiently recovers human T cells from solid tumours, permitting genome-wide CRISPR screens with few mice. Tumour-infiltrating T cells from this model exhibit hallmarks of dysfunction compared with splenic T cells, creating an ideal screening context. We performed two genome-wide CRISPR knockout screens to identify regulators of intratumoural T cell abundance and effector function. The abundance screen revealed the P2RY8-Gα13 GPCR signalling axis as a negative regulator of T cell tumour infiltration. The effector function screen identified GNAS as a key driver of T cell dysfunction in tumours, whose product, Gαs, acts as a convergent node downstream of multiple GPCRs sensing distinct suppressive ligands. Knockout of GNAS rendered T cells resistant to multiple suppressive cues and significantly improved efficacy across diverse solid tumour models in chimeric antigen receptor (CAR) and T cell receptor (TCR) systems. Combinatorial knockout of P2RY8-GNAS further enhanced tumour control, demonstrating that complementary in vivo screens can identify orthogonal targets whose combined editing improves therapeutic potency. This flexible, scalable platform can be adapted for systematic discovery of genetic strategies to improve solid tumour T cell therapies.},
}

Humans
Animals
*T-Lymphocytes/immunology/metabolism/cytology
Mice
*Neoplasms/immunology/genetics/pathology/therapy
*CRISPR-Cas Systems/genetics
GTP-Binding Protein alpha Subunits, Gs/metabolism/genetics/deficiency
*Genome, Human/genetics
Female
Signal Transduction
Lymphocytes, Tumor-Infiltrating/immunology/metabolism
Male
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Receptors, G-Protein-Coupled/metabolism
Gene Knockout Techniques

@article {pmid42642488,
year = {2026},
author = {Yokoyama, K and Chiba, M and Takei, N and Suto, K and Ishio, T and Goto, H and Endo, T and Kadin, ME and Maeda, M and Watanabe, M and Hatakeyama, S and Teshima, T and Yang, Y and Nakagawa, M},
title = {Genome-wide CRISPR screens identify an RXR-MYLIP-LDLR axis regulating BH3 mimetic sensitivity in peripheral T-cell lymphomas.},
journal = {Oncogene},
volume = {45},
number = {41},
pages = {4393-4405},
pmid = {42642488},
issn = {1476-5594},
support = {24K02315//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24K19190//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 23K15314//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; NIH R01 CA259188//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA251674//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; ACS RSG-23-722314//American Cancer Society (American Cancer Society, Inc.)/ ; },
mesh = {Humans ; Animals ; *Receptors, LDL/genetics/metabolism ; Cell Line, Tumor ; *Lymphoma, T-Cell, Peripheral/genetics/drug therapy/pathology/metabolism ; Sulfonamides/pharmacology ; Mice ; CRISPR-Cas Systems ; Aniline Compounds/pharmacology ; Apoptosis/drug effects ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology ; Proprotein Convertase 9/metabolism/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {BH3 mimetics are a promising class of drugs in hematologic malignancies, but their efficacy in peripheral T-cell lymphoma (PTCL) remains poorly understood. To identify genetic determinants of BH3 mimetic response, we performed a genome-wide CRISPR-Cas9 knockout screen in PTCL cell lines and identified MYLIP, an E3 ubiquitin ligase targeting the LDL receptor (LDLR), as a novel sensitizer to navitoclax. MYLIP deletion enhanced the cytotoxicity of navitoclax, venetoclax, and the BCL-XL-selective degrader DT2216 across multiple PTCL models. Mechanistically, MYLIP loss increased LDLR expression, promoted cholesterol uptake, and elevated apoptotic priming. Recombinant PCSK9, which facilitates LDLR degradation, reversed MYLIP knockout-induced sensitization, establishing a functional role for LDLR in BH3 mimetic response. We also identified the nuclear receptor RXRB as an upstream regulator of MYLIP; dual RXRA/RXRB depletion more effectively suppressed MYLIP, upregulated LDLR, and potentiated navitoclax cytotoxicity. In vivo, MYLIP-deficient xenografts showed enhanced tumor suppression with navitoclax treatment. Transcriptomic analyses of primary PTCL samples revealed reduced MYLIP expression in ALK-positive anaplastic large cell lymphoma and genetically defined subsets of nodal T follicular helper cell lymphomas. These findings uncover an RXR-MYLIP-LDLR axis that links cholesterol metabolism to apoptotic susceptibility, offering mechanistic insight into BH3 mimetic sensitivity in PTCL.},
}

Humans
Animals
*Receptors, LDL/genetics/metabolism
Cell Line, Tumor
*Lymphoma, T-Cell, Peripheral/genetics/drug therapy/pathology/metabolism
Sulfonamides/pharmacology
Mice
CRISPR-Cas Systems
Aniline Compounds/pharmacology
Apoptosis/drug effects
Bridged Bicyclo Compounds, Heterocyclic/pharmacology
Proprotein Convertase 9/metabolism/genetics
Xenograft Model Antitumor Assays

@article {pmid42734821,
year = {2026},
author = {Kong, W and Li, J and Wei, R and Wang, S and Li, Y and Pan, W and Li, N and Tang, B},
title = {A steric hindrance-regulated autocatalytic CRISPR/Cas12a system for direct and amplification-free microRNA detection.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {79},
pages = {19840-19844},
doi = {10.1039/d6cc04790d},
pmid = {42734821},
issn = {1364-548X},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {We constructed a CRISPR/Cas12a autocatalytic system based on a miRNA-triggered overhang blocker (OB) cleavage-recombination framework, in which Cas12a sequentially removed steric hindrance to initiate cyclic autocatalysis, enabling exponential signal amplification for visual miRNA detection without target pre-amplification, thereby overcoming direct Cas12a recognition limitations for point-of-care diagnostics.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
Humans
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42818682,
year = {2026},
author = {Luo, Z and Xu, M and Zhang, L and Han, Y and Tan, A and Wang, T and Ye, F and Wang, C and Zhang, J},
title = {Establishment and preliminary evaluation of an RT-ERA combined with CRISPR/Cas12a assay for detection of H5 and H7 influenza viruses.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1922968},
pmid = {42818682},
issn = {2235-2988},
mesh = {Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Influenza A Virus, H5N1 Subtype/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Animals ; DNA Primers/genetics ; *Influenza, Human/diagnosis/virology ; *Hemagglutinin Glycoproteins, Influenza Virus/genetics ; Rapid Diagnostic Tests ; Recombinases/metabolism ; *Influenza A Virus, H7N9 Subtype/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; *Influenza A virus/genetics/isolation & purification/classification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND/OBJECTIVES: Novel influenza A viruses (NIAV) infection is a zoonotic acute respiratory disease. The H5N1 and H7N9 subtypes cause high mortality, posing major public health threats. This study aimed to establish rapid, field-deployable subtype-specific nucleic acid detection assays for H5 and H7 by integrating reverse-transcription enzymatic recombinase amplification (RT-ERA) with CRISPR/Cas12a. Two separate reactions are required per sample.

METHODS: Conserved hemagglutinin (H) sequences were retrieved from the NCBI database, and primers, probes, and crRNAs were designed using Primer Premier 5 and Primer-BLAST. The sensitivity of RT-ERA alone was compared with that of the ERA-CRISPR/Cas12a using serially diluted virus strain nucleic acid. The specificity was verified against other respiratory pathogens with similar clinical manifestations.

RESULTS: The optimal primers were F1R2 for H5 and F2R3 for H7. The limit of detection (LOD) of RT-ERA alone was 2.63×10³ copies/µL for H5 and 1.86×10³ copies/µL for H7; the ERA-CRISPR/Cas12a assay improved the LOD to 2.63×10¹ copies/µL for H5 and 1.86 copies/µL for H7, representing 100-fold and 1000-fold enhancements in sensitivity, respectively. Specificity testing showed that the assay exclusively detected H5N1 and H7N9 with no cross-reactivity to other tested pathogens.

CONCLUSION: The ERA-CRISPR/Cas12a method exhibits enhanced sensitivity for H5 and H7 subtypes detection, along with high specificity, rapidity, and minimal equipment requirements. It offers a promising screening tool for NIAV infections.},
}

Sensitivity and Specificity
Humans
*CRISPR-Cas Systems
*Influenza A Virus, H5N1 Subtype/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Animals
DNA Primers/genetics
*Influenza, Human/diagnosis/virology
*Hemagglutinin Glycoproteins, Influenza Virus/genetics
Rapid Diagnostic Tests
Recombinases/metabolism
*Influenza A Virus, H7N9 Subtype/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
CRISPR-Associated Proteins/genetics
*Influenza A virus/genetics/isolation & purification/classification
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42820720,
year = {2026},
author = {Harakawa, S and Kawakami, H and Kitamura, SI and Ishibashi, N and Watanabe, S and Mekata, T},
title = {Lineage-specific remodeling of a conserved tRNA-Thr-associated genomic island in Edwardsiella anguillarum.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnag119},
pmid = {42820720},
issn = {1574-6968},
abstract = {Edwardsiella anguillarum is a bacterial pathogen affecting diverse aquaculture species worldwide, including red seabream, Japanese eel, and tilapia. In Japan, strains show a phenotypic dichotomy, where red seabream-derived strains are non-motile and ornithine decarboxylase (ODC)-negative, whereas eel-derived strains retain both traits. However, the evolutionary processes underlying lineage diversification within this species remain poorly understood. We performed a global comparative genomic analysis of 24 E. anguillarum strains, including seven newly sequenced genomes from Japan. Despite high ANI values (>99.6%), SNP analysis revealed lineage-level divergence, with 6 500-7 500 SNPs separating red seabream-associated and eel-associated lineages. Comparative analysis identified a conserved chromosomal hotspot adjacent to the tRNA-Thr gene with lineage-specific structural variation. The Japanese red seabream-associated lineage possessed a compact ∼10-kb region containing a Type I-E CRISPR/Cas system, whereas eel-associated and tilapia-derived strains carried expanded genomic islands (∼48-52 kb) enriched in DNA repair and mobile genetic element functions. These configurations were associated with phylogeographic lineages. Genes associated with motility and ODC activity were conserved across lineages, with no evidence of gene loss or major structural disruption. Phylogenetic analysis of the island-associated tyrosine-type recombinase/integrase (YR) was broadly consistent with the core genome. A shared frameshift mutation in eel- and tilapia-derived strains was consistent with a shared pseudogenization event. Overall, our findings indicate that diversification in E. anguillarum is associated with localized structural variation at a conserved tRNA-Thr-associated chromosomal hotspot within an otherwise highly conserved core genome.},
}

@article {pmid42821105,
year = {2026},
author = {Azhar, W and Nasar, I and Fatima, Z and Shaukat, A and Kurniati, TH and Hafiz, I and Murtaza, MA and Irfan, M},
title = {Dynamic, model-guided intensification of fuel ethanol production in yeast: proteome constraints, CRISPR rewiring, and high-gravity fermentations.},
journal = {Archives of microbiology},
volume = {208},
number = {12},
pages = {},
pmid = {42821105},
issn = {1432-072X},
mesh = {*Ethanol/metabolism ; Fermentation ; *Saccharomyces cerevisiae/metabolism/genetics ; Metabolic Engineering/methods ; *Proteome/metabolism/genetics ; Hypergravity ; Biofuels ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The production of fuel ethanol by Saccharomyces cerevisiae is the consequence of the coupling between proteome limitations, metabolism regulation and the final stress of high-gravity fermentation. Recent developments in enzyme constrained genome-scale models and dynamic flux simulations have increased the predictability of the overflow metabolism, pathway usage under stress, and pathways that are critical for growth and ethanol production. Concurrently, CRISPR based genome engineering has permitted the precise modification of the pathways for glycolysis, fermentation, stress tolerance, and pentose sugar metabolism through multiplex editing, promoter tuning, and genome-scale perturbations. Using computational design integrated with CRISPR engineering, machine-learning guided promoter selection, and adaptive laboratory evolution, yeast strains with more balanced metabolic flux and enhanced robustness and ethanol productivity under these challenging growth conditions were obtained. With further advancements in whole-cell model technology, more sophisticated CRISPR tools, synthetic regulatory circuits and AI-based automated engineering platforms, the engineering of robust yeast chassis with improved stability, stress tolerance and productivity for sustainable and large-scale bioethanol generation will be promoted.},
}

*Ethanol/metabolism
Fermentation
*Saccharomyces cerevisiae/metabolism/genetics
Metabolic Engineering/methods
*Proteome/metabolism/genetics
Hypergravity
Biofuels
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42821128,
year = {2026},
author = {Cao, Y and Duan, M and Wei, L and Chen, Y and Liu, F and Bai, J and Huang, Y and Zhang, S},
title = {Serum-compatible aptamer-regulated RCA-CRISPR/Cas12a assay for fluorescence and lateral flow detection of myeloperoxidase.},
journal = {Mikrochimica acta},
volume = {193},
number = {10},
pages = {},
pmid = {42821128},
issn = {1436-5073},
support = {2023QH1706//Fujian Medical University Qihang funds/ ; 2024L3020//the National Filing Science and Technology Project/ ; },
mesh = {*Peroxidase/blood ; Humans ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Fluorescence ; Spectrometry, Fluorescence/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Myeloperoxidase (MPO), a neutrophil-derived heme peroxidase involved in oxidative inflammatory responses, has been investigated as a biomarker for cardiovascular, hematological, and other inflammation-associated diseases. In this study, an aptamer-regulated rolling circle amplification (RCA)-CRISPR/Cas12a fluorescence assay was established for MPO detection in serum samples. Two aptamers, Apt-02 and Apt-14, were obtained with dissociation constants (KD) of 57.76 and 56.88 nM, respectively, using MPO-depleted human serum as the counter-selection matrix during MB-SELEX. Apt-14 exhibited relatively good stability in serum and was therefore used in the assay development. Upon MPO binding, cDNA was released from the aptamer/cDNA duplex, initiating RCA and subsequently activating Cas12a-mediated trans-cleavage to amplify the fluorescence signal. Under the optimized conditions, the assay achieved a detection limit of 0.422 ng/mL with good selectivity. It also showed limited interference from the tested cardiovascular disease-related proteins and provided MPO levels in clinical serum samples that were consistent with ELISA results. The assay was also adapted to a lateral-flow strip using a biotin/FAM-labeled ssDNA reporter, enabling visual detection and reader-assisted semi-quantitative analysis.},
}

*Peroxidase/blood
Humans
*Aptamers, Nucleotide/chemistry/genetics/metabolism
Nucleic Acid Amplification Techniques/methods
Limit of Detection
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Fluorescence
Spectrometry, Fluorescence/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42822620,
year = {2026},
author = {Memariani, H and Memariani, M and Eskandari, SE and Ghasemian, A and KarbalaeiMahdi, A},
title = {Advances in molecular techniques for detecting Mycobacterium leprae: A narrative review of progress, challenges, and future directions.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {594},
number = {},
pages = {122857},
doi = {10.1016/j.cca.2026.122857},
pmid = {42822620},
issn = {1873-3492},
abstract = {Leprosy is a neglected infectious disease where delayed diagnosis leads to disability and ongoing transmission, particularly in paucibacillary and pure neural forms. This narrative review summarizes molecular diagnostic approaches for Mycobacterium leprae, including polymerase chain reaction (PCR), isothermal nucleic acid amplification tests, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based diagnostic systems, next-generation sequencing (NGS), microarrays, and biosensors. PCR remains the most established method, now expanded to multiplex, quantitative, and droplet digital formats for improved detection and antimicrobial resistance surveillance. Isothermal methods such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and multiple cross displacement amplification (MCDA) offer rapid, field-deployable options but require further validation. Emerging platforms, including CRISPR-Cas diagnostics, targeted sequencing, and biosensors, show promise but are limited by cost and infrastructure needs. Overall, molecular approaches have substantially expanded the diagnostic and surveillance capacity for leprosy, particularly in paucibacillary, pure neural, and clinically ambiguous presentations where conventional methods lack sensitivity. Future advances should integrate optimized biomarkers, improved sample processing, and simplified platforms to further enable early diagnosis, treatment monitoring, and antimicrobial resistance surveillance, thereby supporting global leprosy control and elimination.},
}

@article {pmid42823380,
year = {2026},
author = {Szilagyi, Z and Michon, P and Pardo-Hernández, C and Sãcultanu, M and Miralles Fusté, J and Griffin, AM and Charifson, PS and Phan, C and Shi, Y and Kern, G and Martinez Botella, G and Keating, TA and Macao, B and Larsson, NG and Falkenberg, M and Green, J and Gustafsson, CM},
title = {A selective inhibitor reveals roles for LONP1 in mitochondrial proteostasis and OXPHOS dependencies.},
journal = {Life science alliance},
volume = {9},
number = {12},
pages = {},
pmid = {42823380},
issn = {2575-1077},
mesh = {*Mitochondria/metabolism/drug effects ; Humans ; *ATP-Dependent Proteases/metabolism/antagonists & inhibitors/genetics ; *Mitochondrial Proteins/metabolism/antagonists & inhibitors/genetics ; *Proteostasis/drug effects ; *Oxidative Phosphorylation/drug effects ; CRISPR-Cas Systems ; Animals ; Proteomics/methods ; },
abstract = {Lon protease 1 (LONP1) is a conserved hexameric protease implicated in mitochondrial disorders and cancer progression. In this study, we present PZL-26, a potent and selective small-molecule inhibitor that targets LONP1 without affecting the proteasome, leading to selective accumulation of mitochondrial proteins. Using PZL-26 in a whole-genome CRISPR-Cas9 screen, we identified genes essential for cell survival under protease inhibition, supporting a role for LONP1 in key mitochondrial processes, including complex I biogenesis, mitochondrial transcription, and translation. Our CRISPR screen results are consistent with proteomics analysis, with both approaches converging on the same mitochondrial pathways and highlighting functional interactions between LONP1 and other mitochondrial proteases, including potential compensatory mechanisms. These findings establish PZL-26 as an effective tool for exploring LONP1 function and pave the way for future therapeutic strategies targeting LONP1 in mitochondrial diseases and cancer.},
}

*Mitochondria/metabolism/drug effects
Humans
*ATP-Dependent Proteases/metabolism/antagonists & inhibitors/genetics
*Mitochondrial Proteins/metabolism/antagonists & inhibitors/genetics
*Proteostasis/drug effects
*Oxidative Phosphorylation/drug effects
CRISPR-Cas Systems
Animals
Proteomics/methods

@article {pmid42824655,
year = {2026},
author = {Sahu, S and Rajendran, P and Ghumde, SA and Prakash, D and Dixit, S and Pawar, D and Nagaraju, D and T, V and Adavi, SB and Sivalingam, PN and Marathe, A and Rai, PK},
title = {CRISPR/Cas9 in soybean research: a measured leap toward genetic refinement.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1821910},
pmid = {42824655},
issn = {2673-3439},
abstract = {Soybean [Glycine max (Linn.) Merr] is a globally important source of plant-based protein and edible oil, widely cultivated in India as a major oilseed crop. However, conventional breeding approaches for soybean are laborious, constrained by the genomic complexity of soybean, and face persistent challenges in genetic transformation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based genome editing has emerged as a precise and efficient tool for rapid trait improvement in soybean. While many existing reviews of soybean have been published, this literature review addresses some fundamental challenges with respect to genome editing. This study summarizes recent advancements in soybean genome editing, offering insights into the practical informatics of soybean-specific genomic databases that are required for successful construct design. It also discusses emerging technologies such as base editing, prime editing, and the increasingly important role of artificial intelligence in accelerating target identification and construct design. It highlights applications in nutritional enhancement, abiotic and biotic stress tolerance, and improvement of key agronomic traits. Beyond the conventional topics, it addresses the current regulatory landscape that researchers must navigate for releasing genome-edited varieties and provides a comprehensive perspective on practical solutions to overcome major constraints in soybean genetic engineering.},
}

@article {pmid42824670,
year = {2026},
author = {Ning, Y and Lu, C and Wang, Q and Zhao, Y and Wang, Z and Cao, X and Chen, H and Wei, T and Pei, X and Jiang, X and Zhang, F and Chen, Z and Yang, X},
title = {Engineering Kluyveromyces marxianus for efficient 2-phenylethanol production: from metabolic design to process innovation.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1939398},
pmid = {42824670},
issn = {1664-302X},
abstract = {2-Phenylethanol (2-PE) is a valuable aromatic alcohol known for its rose-like scent, widely used in the fragrance, food, and cosmetics industries. The growing demand for "natural" certified products is driving increasing interest in microbial fermentation as a sustainable and promising alternative to chemical synthesis and plant extraction. Kluyveromyces marxianus stands out as a promising microbial host for biomanufacturing due to its thermotolerance, rapid growth, versatile substrate use, and ability to catabolize L-phenylalanine (L-Phe) via the Ehrlich pathway. This review synthesizes recent advances in engineering K. marxianus for competitive 2-PE production, covering both metabolic design and process innovation. It first examines metabolic engineering strategies to build a robust K. marxianus cell factory. These strategies include enhancing central carbon metabolism and precursor biosynthesis, engineering key Ehrlich pathway enzymes to reduce feedback inhibition, optimizing cofactors for redox balance, and improving product tolerance and efflux mechanisms. Looking forward, systems-biology tools-such as multi-omics platforms, genome-scale metabolic models (GSMs), and machine learning-combined with synthetic biology are expected to further rationalize strain design; early applications in K. marxianus (e.g., machine-learning-guided 5' untranslated region optimization) have already been demonstrated, whereas most modeling- and AI-driven strategies for 2-PE production specifically remain at a prospective stage. It also evaluates advances in fermentation process development, including in situ product recovery (ISPR), co-fermentation strategies, and sustainable production from renewable, low-cost feedstocks. By integrating recent progress and analyzing challenges in yield optimization, cellular tolerance, and industrial scalability, this review offers a systematic framework for developing efficient, economically viable, and sustainable 2-PE biomanufacturing processes through the integration of molecular and process-level engineering.},
}

@article {pmid42031623,
year = {2026},
author = {Wang, R and Mao, Y and Guo, Y and Yang, Y and Wei, J and Yang, Y and Zong, S and Zhang, L and Man, S and Peng, W and Ma, L},
title = {Exogenous PAM richer resonator effect unlocks CRISPR/Cas12a biosensor-in-microdroplet for ultrasensitive and multiplexed food safety-related DNA detection.},
journal = {Trends in biotechnology},
volume = {44},
number = {10},
pages = {3080-3103},
doi = {10.1016/j.tibtech.2026.03.024},
pmid = {42031623},
issn = {1879-3096},
mesh = {*Biosensing Techniques/methods ; *Food Safety/methods ; *CRISPR-Cas Systems/genetics ; Food Microbiology ; *DNA, Bacterial/analysis/genetics ; },
abstract = {Accurate, sensitive, and multiplexed DNA detection is crucial for food safety. We describe a counterintuitive phenomenon in which the mere addition of exogenous protospacer adjacent motif richer double-stranded DNA (ePAM-R-dsDNA) boosts the trans-cleavage activity of Lachnospiraceae bacterium Cas12a (LbCas12a) for PAM-lacking dsDNA targets in microdroplets. We call this the exogenous PAM richer resonator effect. Leveraging this, we present a biosensor-in-microdroplet, named CC-Drop-OPT (CRISPR/Cas12a microDroplet Overcoming PAM restriction on Targets), breaking the PAM dependency and highly enhancing (10[4] times) the sensitivity for PAM-lacking dsDNA targets. CC-Drop-OPT demonstrates a wide detection range and a low limit of detection of 10 CFU/ml for foodborne pathogens without DNA preamplification; it is also capable of detecting food authenticity and genetically modified foodstuffs. CC-Drop-OPT supports digital, multiplexed, and background-free identification of three different foodborne pathogens. CC-Drop-OPT introduces a molecular strategy for unamplified DNA detection in food safety and beyond.},
}

*Biosensing Techniques/methods
*Food Safety/methods
*CRISPR-Cas Systems/genetics
Food Microbiology
*DNA, Bacterial/analysis/genetics

@article {pmid42440147,
year = {2026},
author = {Qing, J and Hu, J and Wang, X and Shen, X and Ma, Y and Gao, M and Wu, J},
title = {SnRNA-seq and genome-wide CRISPR screening define the complete transcriptional trajectory and functional drivers of podocyte stress.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42440147},
issn = {1420-9071},
support = {LR26H050001//Natural Science Foundation of Zhejiang Province/ ; 82370717//National Natural Science Foundation of China/ ; },
mesh = {*Podocytes/metabolism/pathology/cytology ; Humans ; *Stress, Physiological/genetics ; *CRISPR-Cas Systems/genetics ; Apoptosis/genetics ; Cell Survival/genetics ; *Glomerulonephritis/genetics/pathology/metabolism ; Transcriptome ; },
abstract = {Podocyte injury is a central driver of proteinuria and progressive kidney dysfunction. Although podocytes are continuously exposed to diverse stressors in both physiological and pathological contexts, the dynamic processes underlying their adaptation and eventual failure remain poorly defined. Here, we performed integrative single-nucleus RNA sequencing of kidney tissues from patients with six types of representative chronic glomerulonephritis, capturing a spectrum of podocyte injury states. We identified distinct podocyte subpopulations and reconstructed a dynamic trajectory characterized by an initial adaptive activation followed by progressive functional decline. Integration with time-resolved transcriptomics identified 778 candidate genes associated with podocyte stress responses. To distinguish putative functional drivers from secondary transcriptional changes, we integrated these candidates with a genome-wide CRISPR-Cas9 knockout screen, prioritizing genes required for podocyte survival under stress conditions. Subsequent siRNA-mediated validation of five representative candidates-BST1, TALDO1, ATP6V1E1, PPP2R1A and CHL1-showed that knockdown of these genes significantly compromised cell viability and accelerated apoptosis, highlighting a coordinated survival network spanning metabolic regulation, autophagy, and cytoskeletal stability. Our findings define a dynamic framework of podocyte stress adaptation and failure, and suggest that targeting stress-response pathways may prolong podocyte survival, thereby extending the therapeutic window for intervention in chronic kidney disease.},
}

*Podocytes/metabolism/pathology/cytology
Humans
*Stress, Physiological/genetics
*CRISPR-Cas Systems/genetics
Apoptosis/genetics
Cell Survival/genetics
*Glomerulonephritis/genetics/pathology/metabolism
Transcriptome

@article {pmid42470099,
year = {2026},
author = {Skeate, JG and Slipek, NJ and Lahr, WS and Roy, S and Wick, BJ and Kar, B and Chang, JW and Stelljes, EM and Gilkey, AK and Thenge, PP and Diers, MD and Krueger, JB and Niemeyer, EM and Lonetree, CL and Kluesner, MG and Bell, JB and Clement, K and Provenzano, PP and Moriarity, BS and Webber, BR},
title = {A singular base-editing platform for polyfunctional multiplex engineering of immune cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {10},
pages = {5858-5871},
doi = {10.1016/j.ymthe.2026.07.033},
pmid = {42470099},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes/immunology/metabolism ; *Receptors, Chimeric Antigen/genetics/metabolism ; *Gene Editing/methods ; Gene Knockout Techniques ; Recombinational DNA Repair ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; Dependovirus/genetics ; Gene Knock-In Techniques ; Genetic Vectors/genetics ; },
abstract = {Current methods to engineer antigen-specific receptors rely on randomly integrating vectors or double-strand-break-induced targeted integration, both of which pose safety risks. To implement an all-in-one tool for multiplex knockout (KO) and knockin (KI), we expand the use of base editor nickase activity to stimulate homology-directed repair (HDR) and insert clinically relevant chimeric antigen receptors (CARs) into specific loci. Through a novel single-guide RNA design strategy and a DNA template delivered by a recombinant adeno-associated virus, we enhanced the efficiency of ABE8e-stimulated HDR in human T cells. By combining KI of CD19, CD33, or mesothelin-targeting CARs with >95% quadplex gene KO (B2M/CD3E/PDCD1/CISH), we achieve single-step generation of highly functional off-the-shelf CAR T cell products with enhanced function. Importantly, we found no detectable translocations or significant off-target edits and demonstrated efficacy against multiple cancer lines and a suppressive 3D spheroid culture model. This efficient engineering process of "Iterative Nicking for Synchronous Engineered Reprogramming of T cells" (INSERT) establishes a safe, simplified platform for advanced therapeutic CAR T engineering.},
}

Humans
*T-Lymphocytes/immunology/metabolism
*Receptors, Chimeric Antigen/genetics/metabolism
*Gene Editing/methods
Gene Knockout Techniques
Recombinational DNA Repair
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods
Dependovirus/genetics
Gene Knock-In Techniques
Genetic Vectors/genetics

@article {pmid42478045,
year = {2026},
author = {Glaser, V and Becker, LJ and Fuster-García, C and Aird, EJ and Huth, L and Nitulescu, AM and Pu, Y and Kassing, I and Hartmann, LM and Flugel, CL and Kārkliņš, R and Shaji, S and Pouzolles, M and Stein, M and Andrieux, G and Corn, JE and Cathomen, T and Volk, HD and Reinke, P and Kath, J and Wagner, DL},
title = {Repurposing base editors for targeted knockin and simultaneous multiplex knockouts to generate allo-CAR T cells with minimal translocations.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {10},
pages = {5872-5889},
doi = {10.1016/j.ymthe.2026.07.034},
pmid = {42478045},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; *T-Lymphocytes/metabolism/immunology ; CRISPR-Cas Systems ; Humans ; *Gene Knock-In Techniques/methods ; *Translocation, Genetic ; Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/metabolism ; Transgenes ; DNA Breaks, Double-Stranded ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Animals ; },
abstract = {Multiplex genome editing of cellular therapies frequently requires multiple DNA double-strand breaks (DSBs), which can induce genotoxicity through chromosomal rearrangements and large deletions. Base editors enable targeted sequence changes with minimal DSBs and are widely used for gene disruption, but their capacity for transgene insertion has remained unexplored. Here, we have developed base editor-mediated knockin (BEKI), a non-viral platform combining transgene insertion with multiplex gene disruption using a single enzyme. BEKI repurposes the base editor's Cas9 nickase domain to generate paired nicks (inducing a localized DSB) at the knockin locus while achieving multiplex knockouts through base editing. Optimized guide RNA orientation and spacing enabled efficient transgene insertion across multiple T cell-relevant genomic loci. DNA-PK inhibition enhanced knockin efficiency but increased kilobase-scale deletions, which were mitigated by co-inhibition of Polθ. Compared with multiplex Cas9 editing, BEKI markedly reduced chromosomal translocations while preserving cell viability. BEKI supported targeted chimeric antigen receptor (CAR) knockin alongside up to 10 simultaneous gene knockouts, enabling the generation of allogeneic CAR T cells with enhanced cytokine secretion and resistance to immunosuppressants and allo-rejection. Together, BEKI provides a streamlined and scalable strategy for multiplex CAR T cell engineering with improved genomic stability, advancing safer next-generation cell therapies for cancer and autoimmune diseases.},
}

*Gene Editing/methods
*T-Lymphocytes/metabolism/immunology
CRISPR-Cas Systems
Humans
*Gene Knock-In Techniques/methods
*Translocation, Genetic
Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/metabolism
Transgenes
DNA Breaks, Double-Stranded
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockout Techniques
Animals

@article {pmid42528134,
year = {2026},
author = {Zhao, D and Zhao, X and Gao, Y and Yang, S and Guo, X and Zhang, C and Li, C and Zhang, C and Tian, H and Xu, Q and Yin, Z and Liu, Z and Song, J},
title = {A helicase-fused Cas9 improves large-size fragment knockin.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {10},
pages = {6153-6162},
doi = {10.1016/j.ymthe.2026.07.049},
pmid = {42528134},
issn = {1525-0024},
mesh = {Humans ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; Homologous Recombination ; Rabbits ; *Recombinant Fusion Proteins/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; Cell Line ; Gene Editing/methods ; Minichromosome Maintenance Proteins/genetics ; Cell Cycle Proteins ; },
abstract = {Homologous recombination (HR)-mediated large-size fragment knockin (ls-KI) remains inefficient in mammalian cells, even with the assistance of CRISPR-Cas9. We hypothesized that adding DNA helicase activity to CRISPR-Cas9 could enhance ls-KI efficiency. To test this, we fused MCM5, a subunit of the eukaryotic MCM2-7 helicase complex, to the N terminus of spCas9. The resulting fusion protein, termed MCCas, markedly increased knockin rates across multiple loci in different human cell lines, significantly outperforming spCas9. Remarkably, MCCas enabled efficient ls-KI with donor templates of up to 10 kb in size. MCCas-mediated ls-KI, in comparison to those mediated by spCas9, is also associated with reduced frequencies of on-target and off-target insertion and deletion (indel) events. Mechanistic investigations revealed that MCCas-mediated ls-KI relies on the canonical HR pathway, as inhibition of key processes such as end resection and strand invasion abolished the enhancement. To further validate its application, we employed MCCas to knock in the human ACE2 (hACE2) coding sequence to the rabbit genome. Consistent with our findings in human cells, MCCas led to a more than 2-fold increase in ls-KI rates in rabbit embryos compared to spCas9. Collectively, our results establish MCCas as a promising gene-editing tool with enhanced ls-KI capacity.},
}

Humans
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems
Animals
*CRISPR-Associated Protein 9/genetics/metabolism
Homologous Recombination
Rabbits
*Recombinant Fusion Proteins/genetics/metabolism
*DNA Helicases/genetics/metabolism
Cell Line
Gene Editing/methods
Minichromosome Maintenance Proteins/genetics
Cell Cycle Proteins

@article {pmid42614060,
year = {2026},
author = {Feng, S and Li, Y and Ye, T and Su, P and Li, X and Wang, W},
title = {A surface-mediated hydrogel patch for localized CRISPR genome editing in inflammatory wound therapy.},
journal = {Materials horizons},
volume = {13},
number = {19},
pages = {9834-9846},
doi = {10.1039/d6mh01100d},
pmid = {42614060},
issn = {2051-6355},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Hydrogels/chemistry ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics ; *CRISPR-Cas Systems ; Staphylococcus aureus ; Wound Healing ; Inflammation/therapy ; Staphylococcal Infections/therapy ; Cell Line ; },
abstract = {The NLRP3 inflammasome is a central driver of inflammatory tissue injury and delayed repair in bacteria-infected wounds, yet local genome editing in such lesions remains limited by nonspecific release and off-target exposure. Here, we report a pH-gated surface-mediated hydrogel patch for infection-responsive delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes. Rather than embedding the cargo within the bulk matrix, polymer-coated NLRP3-targeting RNPs are reversibly anchored on the hydrogel surface, enabling localized presentation under physiological conditions and rapid detachment in the acidic microenvironment of Staphylococcus aureus (S. aureus)-infected wounds. In vitro studies demonstrated an NLRP3 editing efficiency of 57% in J774A.1 cells and 69% in L929 cells surpassing or matching the performance of the commercial transfection reagent Lipofectamine CRISPRMAX (50% in J774A.1; comparable in L929). In a murine full-thickness skin defect model with S. aureus infection, topical administration of the patch achieved >20% NLRP3 editing efficiency in wound tissue, accompanied by a statistically significant reduction in inflammatory cell infiltration. Consequently, this pH-responsive surface anchoring and release strategy establishes a novel paradigm for localized, precision gene therapy and holds strong translational promise for the treatment of chronic inflammatory wounds.},
}

Animals
Mice
*Gene Editing/methods
*Hydrogels/chemistry
NLR Family, Pyrin Domain-Containing 3 Protein/genetics
*CRISPR-Cas Systems
Staphylococcus aureus
Wound Healing
Inflammation/therapy
Staphylococcal Infections/therapy
Cell Line

@article {pmid42625514,
year = {2026},
author = {Carballar-Lejarazú, R and McNeely, B and Dong, Y and Pham, TB and Tushar, T and Owusu-Frimpong, I and Dimopoulos, G and James, AA},
title = {Impact of naturally occurring target-site polymorphisms on an autonomous Cas9/guide RNA-based gene drive system for population modification of Anopheles gambiae s.l.},
journal = {Genetics},
volume = {234},
number = {2},
pages = {},
doi = {10.1093/genetics/iyag221},
pmid = {42625514},
issn = {1943-2631},
support = {//University of California Irvine Malaria Initiative/ ; NIAID R01 AI170692//National Institute of Allergy and Infectious Diseases/ ; INV-043645//Bill & Melinda Gates Foundation/ ; //Bloomberg Philanthropies/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; Mosquito Vectors/genetics ; Malaria/prevention & control/transmission ; },
abstract = {Malaria remains a significant global health challenge with an estimated 282 million cases reported in 2024. CRISPR/Cas9-based gene-drive systems have emerged as promising tools to block Plasmodium transmission by mosquito vectors. The TP13 drive system targets the Anopheles gambiae cardinal (Agcd) gene and carries 2 engineered monoclonal antibodies to achieve rapid population modification to prevent parasite transmission. Previous cage trials demonstrated complete drive introduction in 3 to 6 generations and supported modeling predicting a potential >90% reduction in malaria incidence under optimal conditions. However, naturally-occurring genetic polymorphisms in wild mosquito populations, particularly single-nucleotide polymorphisms (SNPs) within Cas9/guide RNA target sites, pose a potential barrier to drive efficiency. High genetic diversity in An. gambiae results in drive-system target-site variants, including an A→T transversion in the Agcd gene, which occurs at high frequencies in African populations and could affect TP13 drive dynamics. The impact of this and other SNPs on TP13 performance were assessed by establishing 3 An. gambiae Ndokayo lines, 1 with the wild-type Agcd and 2 with homozygous SNP haplotypes. We evaluated drive conversion rates in vivo, population dynamics in cage trials, fitness costs, and parasite suppression efficacy. No negative effects on drive performance and parasite suppression were observed. The results provide insights into the influence of naturally-occurring polymorphisms on gene drive propagation, informing safety, efficacy, and target product profile requirements for advancing gene-drive mosquitoes toward field trials.},
}

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

@article {pmid42691137,
year = {2026},
author = {Duarte, VN and Najafi, V and Thompson-Peer, KL},
title = {Optimizing CRISPR/Cas9 mutagenesis in Drosophila dendritic arborization neurons to avoid cytotoxicity.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkag211},
pmid = {42691137},
issn = {2160-1836},
support = {NIH-S10OD032327-01//Cancer Center Support/ ; //UCI School of Biological Sciences ODEI Postdoctoral Excellence/ ; //Optical Biology Core Facility of the Developmental Biology Center/ ; CA-62203//Cancer Center Support/ ; //Vienna Drosophila Resource Center/ ; //Hellman Family Foundation/ ; S10 OD032327/OD/NIH HHS/United States ; //Rose Hills Foundation/ ; T32 MH119049/MH/NIMH NIH HHS/United States ; T32MH119049//University of California, Irvine/ ; R00 NS097627/NS/NINDS NIH HHS/United States ; NIH P40OD018537//Bloomington Drosophila Stock Center/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Mutagenesis ; *Neurons/metabolism/cytology ; *Dendrites/metabolism ; Transgenes ; *Drosophila melanogaster/genetics ; },
abstract = {Genetic perturbations are one of the great strengths of the model organism Drosophila melanogaster, with approaches such as classical mutagenesis and RNA interference enabling a wealth of biological discoveries. A more recent approach for altering gene expression is CRISPR/Cas9-based mutagenesis. As with any new tool, however, its use must be optimized. High expression of Cas9 has been shown to cause cytotoxicity in some cell types, including class IV dendritic arborization (da) neurons. In this study, we provide evidence that Cas9 expression causes cytotoxicity in class I da neurons, in addition to class IV da neurons, both of which are widely used to study neuronal development and regeneration. We then systematically evaluated available Cas9 transgenes designed to titrate Cas9 expression, called uCas9 transgenes. We show that the expression of these uCas9 transgenes results in little to no cytotoxicity in various classes of da neurons. Immunostaining revealed drastic reductions in Cas9 protein levels for da neurons expressing the uCas9(L) transgene. Lastly, we demonstrate that the uCas9(L) transgene effectively and specifically gene edits in both class I and class IV da neurons, lowering the expression of GFP-tagged proteins and producing loss-of-function morphological phenotypes when targeting endogenous loci. Thus, we refine the use of CRISPR mutagenesis in Drosophila da neurons through titration of Gal4/UAS-mediated Cas9 expression using existing uCas9 transgenes, a feasible and flexible approach that may be useful for other labs encountering Cas9 cytotoxicity in their own model systems.},
}

Animals
*CRISPR-Cas Systems
*Mutagenesis
*Neurons/metabolism/cytology
*Dendrites/metabolism
Transgenes
*Drosophila melanogaster/genetics

@article {pmid42714387,
year = {2026},
author = {Mukherjee, A and Assefa, AB and Turlo, CV and Needham, LC and Shoue, D and Qahash, T and Belachew, M and Tadesse, D and Kassie, E and Berihun, M and Brhane, BG and Parr, JB and Ferdig, MT and , },
title = {Functional validation of the Plasmodium falciparum K13 C580Y mutation in recently collected Ethiopian isolates.},
journal = {Antimicrobial agents and chemotherapy},
volume = {70},
number = {10},
pages = {e0049526},
doi = {10.1128/aac.00495-26},
pmid = {42714387},
issn = {1098-6596},
support = {R01AI177791//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects/isolation & purification ; Artemisinins/pharmacology ; *Antimalarials/pharmacology ; Ethiopia ; Drug Resistance/genetics ; Mutation/genetics ; Humans ; Malaria, Falciparum/parasitology/drug therapy ; *Protozoan Proteins/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Recent genomic investigation in Ethiopia identified the first detection of the Plasmodium falciparum Kelch13 (K13) C580Y substitution in the Horn of Africa. To assess its functional impact, we introduced C580Y into two recently collected Ethiopian clinical isolates using CRISPR-Cas9 genome editing. Ring-stage survival assays showed significantly elevated in vitro dihydroartemisinin survival in edited parasites relative to isogenic controls, demonstrating that C580Y confers artemisinin tolerance in contemporary Ethiopian parasite genetic backgrounds.},
}

*Plasmodium falciparum/genetics/drug effects/isolation & purification
Artemisinins/pharmacology
*Antimalarials/pharmacology
Ethiopia
Drug Resistance/genetics
Mutation/genetics
Humans
Malaria, Falciparum/parasitology/drug therapy
*Protozoan Proteins/genetics
CRISPR-Cas Systems/genetics

@article {pmid42816508,
year = {2026},
author = {Brodmann, M and Baca, CF and Chandanani, J and Campbell, EA and Marraffini, LA},
title = {MtvS1 and MtvS2 Interact with RNA polymerase to regulate the Francisella type V-A CRISPR-Cas system.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42816508},
issn = {2041-1723},
support = {R35 GM151879/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacterial Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Francisella/genetics/metabolism/pathogenicity ; *DNA-Directed RNA Polymerases/metabolism/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Bacteria and archaea often harbor multiple CRISPR-Cas loci to defend against mobile genetic elements. Little is known, however, about whether and how different CRISPR-Cas systems are differentially regulated, in many instances due to the impossibility of studying CRISPR immunity in native hosts. Here we investigated the regulation of the endogenous type II-B and type V-A CRISPR-Cas systems present in the opportunistic human pathogen Francisella novicida U112. We found that while the type II-B system is constitutively expressed, the type V-A system is differentially expressed at stationary phase and high cell density. We identified MtvS1 and MtvS2 as factors required for this regulation, as well as for the modulation of many additional genes in stationary phase, some of which are required for Francisella virulence. Both Francisella MtvS proteins bind to RNA polymerase. MtvS1 is predicted to interact with the β' subunit of the RNA polymerase, and MtvS2 with multiple RNA polymerase subunits as well as MtvS1. We propose that MtvS1 and MtvS2 constitute noncanonical alternative sigma factors involved in the regulation of the expression of the type V-A CRISPR locus and other genes in Francisella. Last, we show that the MtvS1 homolog YgfB is required for expression of the type I-E CRISPR-Cas system in E. coli, a result that suggests a broader role in gene regulation for these alternative sigma factors.},
}

*Bacterial Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*Francisella/genetics/metabolism/pathogenicity
*DNA-Directed RNA Polymerases/metabolism/genetics
Gene Expression Regulation, Bacterial

@article {pmid42816914,
year = {2026},
author = {Sponchiado, M and Mangiavacchi, P and Redel, BK and Lee, K},
title = {Precision Genome Editing in Domestic Animals: A New Era for Agriculture and Medicine.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {61 Suppl 3},
number = {},
pages = {e70286},
doi = {10.1111/rda.70286},
pmid = {42816914},
issn = {1439-0531},
support = {//National Institute of Allergy and Infectious Disease/ ; //National Heart, Lung and Blood Institute/ ; U42OD011140/OD/NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/veterinary/methods ; Animals, Genetically Modified/genetics ; Agriculture/methods ; *Livestock/genetics ; Swine/genetics ; CRISPR-Cas Systems ; Genetic Engineering/veterinary ; Animals, Domestic/genetics ; },
abstract = {Precision genome editing technologies have transformed the generation and application of genetically engineered livestock, positioning domestic animals as powerful models for agriculture, biomedicine, and basic research. Historically, genetic engineering in livestock was constrained by the absence of embryonic stem cells, low efficiency of homologous recombination, long gestation periods, and extended generation intervals. The advent of programmable nucleases, including zinc finger nucleases, transcription activator-like effector nucleases, and CRISPR-Cas systems, has overcome many of these barriers by enabling efficient targeted genome modification. This review focuses on the application of genome editing technologies in mammalian livestock species, with pigs highlighted as a representative model. We discuss the historical development of livestock genetic engineering and summarise two main routes used to establish genome edited livestock models. Key technical considerations unique to domestic animals are also presented. Genome editing has accelerated the production of livestock models for human disease, xenotransplantation, reproductive biology, and agricultural improvement, including disease resistance, altered growth traits, and enhanced product composition. Emerging epigenome editing strategies further expand the toolkit by allowing locus-specific gene regulation without permanent DNA modification. Despite rapid technological progress, challenges remain, including mosaicism, off-target effects, limited ability to segregate unintended edits, and ethical and regulatory concerns surrounding food and biomedical applications. Continued refinement of genome editing strategies will be essential for maximizing the utility and responsible implementation of genome-edited livestock. Overall, precision genome editing represents a transformative platform with broad implications for sustainable agriculture, translational research, and global food security.},
}

Animals
*Gene Editing/veterinary/methods
Animals, Genetically Modified/genetics
Agriculture/methods
*Livestock/genetics
Swine/genetics
CRISPR-Cas Systems
Genetic Engineering/veterinary
Animals, Domestic/genetics

@article {pmid42817081,
year = {2026},
author = {Pushp, and Paliwal, A},
title = {Fungal Keratitis: A Narrative Review with a Focus on Antifungal Resistance and Emerging Therapies.},
journal = {Infectious disorders drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118715265490828260921074616},
pmid = {42817081},
issn = {2212-3989},
abstract = {Corneal infection that can cause blindness, fungal keratitis has shown an increasing prevalence worldwide, especially in tropical and subtropical areas, where it significantly increases the risk of unilateral corneal blindness. The epidemiology, molecular etiology, diagnosis, antifungal resistance, and changing treatment of fungal keratitis are all reviewed in this article. Geographic diversity is evident in epidemiological patterns, with filamentous fungi such as Aspergillus and Fusarium predominating in warm climes, and an increasing number of reports of Candida species in temperate and healthcare-associated settings. Disease incidence and outcomes have been further affected by post-COVID-19 changes in ophthalmic treatment, extensive corticosteroid exposure, delayed presentations, and environmental factors, including climate fluctuations and increased airborne spore loads. Rapid stromal invasion, enzyme-mediated tissue damage, biofilm development, and host immune dysregulation are key factors determining severity, according to molecular findings. For improved detection, including in culture-negative and polymicrobial cases, diagnostic advancements focus on combining traditional microscopy and culture with in vivo confocal microscopy, MALDI-TOF MS, targeted PCR, and metagenomic next-generation sequencing. Time to treatment is being reduced by rapid point-of-care techniques like tear β-D-glucan tests, LAMP, CRISPR/Cas platforms, and AI-based image analysis. The necessity for systematic susceptibility testing and antifungal management is supported by rising antifungal resistance, particularly rising MIC trends in Fusarium and Aspergillus species, which exhibit variable susceptibility to azoles and amphotericin B, highlighting the need for species-specific antifungal susceptibility testing. The evolving epidemiology, molecular etiology, and new developments in fungal keratitis diagnosis are covered in this article. Additionally, it emphasizes new therapeutic approaches, including advanced drug delivery systems, advances in antifungal resistance management, and potential avenues for improved management. The article presents a narrative review focusing on recent developments in the epidemiology, molecular mechanisms, diagnosis, antifungal resistance, and treatment options for fungal keratitis.},
}

@article {pmid42818019,
year = {2026},
author = {Villani, A and Karambelkar, S and Pathak, N and Kamath, R and Govindarajan, S and Csörgő, B and Varnavski, NR and Bondy-Denomy, J},
title = {A ΦKMV ligase-dependent DNA repair mechanism that mitigates DNA-targeting nucleases.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.09.21.753257},
pmid = {42818019},
issn = {2692-8205},
abstract = {UNLABELLED: Bacteria employ diverse DNA-targeting systems, including restriction-modification (R-M) and CRISPR-Cas, to cleave invading bacteriophage genomes. In response, phages encode counter-defense strategies that block or mitigate DNA damage. Here, we screened a panel of Pseudomonas aeruginosa phages against native and heterologous DNA-targeting systems and identified the Phikmvvirus phage genus as broadly resistant to multiple CRISPR-Cas and R-M systems. Following CRISPR-Cas12a exposure, most protospacer sequences remained genetically unchanged. However, at an intergenic protospacer, mutations accumulated with high frequency at the Cas12a cleavage site rather than within PAM or seed sequences, resembling repair-associated indels observed after genome editing in eukaryotic cells. Genetic screens to isolate Cas12a- and EcoRI-sensitized phage mutants revealed perturbations to the phage DNA ligase. A Cas12a-sensitive mutant phage was rescued by DNA ligase expression in trans, which was also sufficient to reverse CRISPR targeting of an unrelated phage. Together, our results support a model in which ΦKMV-like phages tolerate certain nucleases through ligase-dependent repair of nuclease-induced double-stranded breaks.

IMPORTANCE: Bacterial resistance to antimicrobial medication is escalating, and yet new antibiotics are not readily available. Without novel antibiotics, phage therapy has emerged as a viable response to the antibiotic resistance. Ideally, phage will achieve broad host range through layered anti-defense strategies that ensure their replicative success. Here we describe a broad-acting mechanism that allows Phikmvvirus phages to evade nuclease targeting. Through a phage encoded DNA ligase, gp17, ΦKMV phage seems to repair at predicted cut sites, often with high fidelity but occasionally leaving scars reminiscent of NHEJ repair. Active phage DNA ligases also support nuclease evasion by a distinct phage, DMS3. These findings describe phage escape through faithful repair and identify a potentially interesting gene for phage therapy.},
}

@article {pmid42818483,
year = {2026},
author = {Nureki, O and Onishi, K and Shuto, Y and Müller, J and Shibata, M and Omura, S},
title = {Visualizing structural dynamics during nuclease activation across evolutionarily diverse IscB and CRISPR-Cas9 enzymes.},
journal = {Research square},
volume = {},
number = {},
pages = {},
doi = {10.21203/rs.3.rs-10202707/v1},
pmid = {42818483},
issn = {2693-5015},
abstract = {Cas9 nucleases of CRISPR-Cas adaptive immune systems are programmable RNA-guided DNA endonucleases that evolved from the transposon-associated IscB enzymes. Comparative structural studies have revealed substantial architectural elaboration during this evolutionary transition, including the replacement of the larger ωRNA scaffold of IscB with the REC domains of Cas9, capable of recognizing longer guide-target heteroduplexes. However, these evolutionary insights remain restricted to static structural features, and the dynamic properties that underlie nuclease activation have not been systematically explored across the IscB-to-Cas9 lineage. Here, by combining cryo-electron microscopy structural analysis with real-space, real-time high-speed atomic force microscopy imaging, we visualize the stepwise nuclease activation mechanisms of evolutionarily diverse IscB and Cas9 nucleases, including IscB.m13, type II-B PsCas9, type II-C CjCas9, and type II-D Cas9d MG34-1. We show here that the IscB family employs two distinct strategies for HNH autoinhibition, with one representing the direct evolutionary precursor of the Cas9 nuclease activation mechanism. Moreover, although the stacking-based protein-DNA interaction that stabilizes an intermediate state along the activation pathway is conserved in both IscB and Cas9 enzymes, Cas9 has acquired an additional gating element that prevents premature HNH activation immediately before catalysis. Together with previously reported structures of OgeuIscB and SpCas9, these analyses establish an atlas of the structural dynamics underlying nuclease activation across the IscB-to-Cas9 lineage, uncovering both conserved and diversified activation mechanisms and substantially advancing the mechanistic and evolutionary understanding of these RNA-guided endonucleases.},
}

@article {pmid42679594,
year = {2026},
author = {Wen, W and Li, X and Liu, T and Zhang, SM and Sun, D and Zhao, XD and Cheng, CY and Zhang, LM and Zhang, JP and Zhang, XB},
title = {Sequential CD2/CD3/CD28 activation improves nonviral large-payload chimeric antigen receptor knock-in at the T cell receptor α constant locus.},
journal = {Cytotherapy},
volume = {28},
number = {11},
pages = {102955},
doi = {10.1016/j.jcyt.2026.102955},
pmid = {42679594},
issn = {1477-2566},
mesh = {Humans ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *CD3 Complex/immunology/genetics/metabolism ; *CD28 Antigens/immunology/metabolism/genetics ; Mice ; Gene Knock-In Techniques/methods ; *CD2 Antigens/immunology/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Lymphocyte Activation ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; },
abstract = {CRISPR/Cas9-mediated nonviral targeted integration via homology-directed repair (HDR) offers a strategy for generating uniform, next-generation chimeric antigen receptor (CAR) T cells while avoiding risks associated with viral vectors. However, efficient delivery of large therapeutic payloads into primary T cells is limited by low HDR efficiency and electroporation-induced toxicity. Here, we show that sequential CD2/CD3/CD28 activation improves HDR-mediated large-payload knock-in at the T cell receptor α constant (TRAC) locus and increases viable CAR-T cell yield under an AZD7648-supported nonviral editing framework. Transcriptomic profiling and mitochondrial membrane potential analysis suggest that this condition is associated with cell-cycle- and metabolism-related programs, which may contribute to improved tolerance to electroporation stress and processing of large DNA templates. Functionally, engineered nonviral TRAC-CD19.CAR-T cells exhibit antigen-specific cytotoxicity in vitro and suppress leukemia progression in an NSG xenograft model, with antitumor activity that approached that of a lentiviral CAR-T reference in this proof-of-function xenograft model. This work establishes an optimized sequential activation strategy to improve nonviral, large-payload TRAC-targeted CAR-T cell engineering and may inform future development of precision cellular immunotherapy manufacturing workflows.},
}

Humans
Animals
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*CD3 Complex/immunology/genetics/metabolism
*CD28 Antigens/immunology/metabolism/genetics
Mice
Gene Knock-In Techniques/methods
*CD2 Antigens/immunology/genetics/metabolism
*Immunotherapy, Adoptive/methods
T-Lymphocytes/immunology
Lymphocyte Activation
CRISPR-Cas Systems
Xenograft Model Antitumor Assays

@article {pmid42679634,
year = {2027},
author = {Chen, W and Fang, J and Ran, M and Wang, L and Cui, S and Chen, Q and Cui, J and Chen, Y and Wang, J and Lu, C and Jiang, H},
title = {Rapid on-site and simultaneous detection of four foodborne Vibrio species in seafood using a microfluidic RPA-CRISPR/Cas12a platform.},
journal = {Talanta},
volume = {312},
number = {Pt C},
pages = {130547},
doi = {10.1016/j.talanta.2026.130547},
pmid = {42679634},
issn = {1873-3573},
mesh = {*Seafood/microbiology ; *Vibrio/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; Rapid Diagnostic Tests ; Vibrio parahaemolyticus/isolation & purification/genetics ; Animals ; Lab-On-A-Chip Devices ; Limit of Detection ; },
abstract = {Foodborne Vibrio species commonly contaminate seafood and often co-occur, complicating rapid on-site detection. We developed a centrifugal microfluidic chip integrating one-pot RPA-CRISPR/Cas12a for simultaneous detection of four key species: V. parahaemolyticus, V. vulnificus, V. cholerae, and V. alginolyticus. In this system, target-specific RPA amplification generates amplicons that activate Cas12a-mediated trans-cleavage of fluorescent ssDNA reporters, producing target-dependent fluorescence signals for pathogen identification. The chip has eight units, each with four reaction chambers with lyophilized target-specific reagents, enabling a simple "DNA-in, result-out" workflow. Operating at 39 °C, the assay is completed within 60 min in a closed system to minimize contamination. It shows high specificity with no cross-reactivity. Limits of detection are 10[0] copies/μL for V. parahaemolyticus and V. vulnificus, and 10[1] copies/μL for V. cholerae and V. alginolyticus. Performance in spiked and real shrimp samples matched qPCR, with 100% sensitivity and specificity. The portable platform costs about $3.29 per test. This platform offers a practical approach for on-site multiplex screening of pathogenic Vibrio in seafood.},
}

*Seafood/microbiology
*Vibrio/isolation & purification/genetics
*CRISPR-Cas Systems
*Food Microbiology/methods
Rapid Diagnostic Tests
Vibrio parahaemolyticus/isolation & purification/genetics
Animals
Lab-On-A-Chip Devices
Limit of Detection

@article {pmid42696954,
year = {2027},
author = {Zhang, J and Liu, G and Yu, S and Chen, Z and Yuan, Z and Zhang, J and Hu, J and Diao, Y and Tang, Y and He, D},
title = {A single-tube dual-channel CRISPR platform for genotyping of waterfowl reoviruses.},
journal = {Talanta},
volume = {312},
number = {Pt C},
pages = {130574},
doi = {10.1016/j.talanta.2026.130574},
pmid = {42696954},
issn = {1873-3573},
mesh = {Animals ; Genotype ; *Orthoreovirus, Avian/genetics/isolation & purification ; Ducks/virology ; *Genotyping Techniques/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Reoviridae Infections/veterinary/virology/diagnosis ; *CRISPR-Cas Systems ; },
abstract = {Waterfowl reovirus (WRVs) includes two genotypes, Genotype I (Muscovy duck reovirus, MDRV) and Genotype II (novel duck reovirus, NDRV). They have the same clinical manifestations, but different pathogenicity, and can co-infect individual hosts, resulting in diagnostic ambiguity that cannot be solved by single-target detection. Here, we report a single-tube dual-channel platform coupling recombinase polymerase amplification (RPA) with orthogonal CRISPR effectors: LbCas12a targeting the conserved S4 gene of MDRV via a ROX-labeled ssDNA reporter, and LwaCas13a targeting the S1 gene of NDRV via a FAM-labeled ssRNA reporter. The integrated format enables simultaneous genotypic differentiation within 30 min at isothermal temperatures, producing exclusive ROX-channel signal for MDRV, exclusive FAM-channel signal for NDRV, and concurrent dual-channel signal for co-infected samples, with no cross-channel interference (p > 0.05). The assay achieved a limit of detection of 1 copy/μL for both targets with no cross-reactivity against four co-circulating waterfowl pathogens. An ImageJ-based RGB quantification standard enables equipment-minimal visual classification of all four diagnostic outcomes. Clinical evaluation of 133 field specimens yielded complete concordance (100%) with qPCR, correctly identifying 15 MDRV-positive, 67 NDRV-positive, 8 co-infected, and 43 negative cases. This platform provides a validated, equipment-minimal tool for concurrent WRV genotyping and field surveillance.},
}

Animals
Genotype
*Orthoreovirus, Avian/genetics/isolation & purification
Ducks/virology
*Genotyping Techniques/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Reoviridae Infections/veterinary/virology/diagnosis
*CRISPR-Cas Systems

@article {pmid42802910,
year = {2026},
author = {Zhou, J},
title = {Addressing the Blind Spots of Deaminase Base Editors with CRISPR-Cas Glycosylase-Based Editors.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {25731599261475668},
doi = {10.1177/25731599261475668},
pmid = {42802910},
issn = {2573-1602},
abstract = {The emergence of CRISPR base editors signifies a pivotal shift in genome editing, moving beyond the "cut-and-paste" approach into an era of precise "chemical rewriting." While early editors including CBEs and ABEs enabled efficient base transitions by combining deaminases with Cas9 nickase, their core mechanism limited their application to transition mutations, leaving nearly half of disease-causing transversions unresolved. This review outlines the transformation of DNA glycosylases from unwanted side-effect generators into core drivers of base editor evolution. Through strategic protein engineering, glycosylases have been harnessed to create novel editors that mediate crucial transversion edits, substantially expanding the targeting landscape of precision genome editing. We will examine how these engineered glycosylases achieve programmable DNA rewriting through redirected DNA repair pathways, compare emerging and conventional editors, and explore applications in genetic therapy and functional genomics while addressing ongoing challenges in specificity and delivery. Finally, we envision next-generation editors equipped with artificial intelligence-designed glycosylases capable of spatiotemporally controlled genome manipulation.},
}

@article {pmid42805175,
year = {2026},
author = {Song, D and Malinao, MG and Wu, X and Jiang, S and Jiang, D and Westberg, M and Su, Y and Cai, S and Lee, D and Repina, NA and Schaffer, DV and Ji, HP and Hong, G and Lin, MZ},
title = {Non-invasive control of gene editing in vivo by photoswitchable Cas12f and focused ultrasound.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2026.09.005},
pmid = {42805175},
issn = {1097-4172},
abstract = {A spatiotemporally controllable CRISPR-Cas system would be highly desirable for restricting gene editing to specific times and locations in vivo. Ideally, such a system would be compatible with tissue- or cell-type-specific delivery by adeno-associated virus (AAV) and could be controlled from outside the body. However, neither the delivery of controllable CRISPR-Cas systems by single AAV particles nor their noninvasive activation deep within tissue has yet been achieved. In this study, we address both of these limitations. We engineer compact photoswitchable Cas12f (psCas12f) proteins that are co-deliverable with their single guide RNA (sgRNA) in a single AAV particle. We then activate psCas12f in vivo using focused ultrasound (FUS) and mechanoluminescent particles, enabling precise spatiotemporal control. We demonstrate successful gene editing in the targeted skeletal muscle and brain regions of mice using AAV-transduced psCas12f and FUS. In summary, we describe a completely noninvasive method for localized gene editing in vivo.},
}

@article {pmid42805377,
year = {2026},
author = {Xie, Z and Oh, H and Jin, YS and Miller, MJ},
title = {Restoration of lactose metabolism in Lacticaseibacillus rhamnosus GG for dairy fermentation applications.},
journal = {Journal of dairy science},
volume = {},
number = {},
pages = {},
doi = {10.3168/jds.2026-29059},
pmid = {42805377},
issn = {1525-3198},
abstract = {Lacticaseibacillus rhamnosus GG (LGG) is a widely used probiotic but has limited applications in dairy fermentation because of its poor lactose utilization. Here, we generated a lactose-positive LGG derivative using the LGG endogenous CRISPR-Cas system achieving a marker-free, scarless 20-bp deletion to disrupt the t1 transcriptional terminator upstream of the lactose operon. Using lactose-based selection, lactose-positive derivatives were then isolated and confirmed a restored functional lacG allele by sequencing. After curing the editing plasmid, the final strain, MJM570, was obtained. Whole-genome sequencing verified the intended terminator deletion and lacG reversion. MJM570 grew robustly on lactose, supported growth and acidification in milk, and showed improved proliferation and viability in yogurt fermentation and refrigerated storage compared with the wild-type LGG. This marker-free strategy leaves no foreign DNA in the final strain and provides a practical platform for developing dairy-compatible probiotic cultures with improved performance in fermented dairy applications. Overall, this study demonstrates the potential of endogenous CRISPR-Cas systems for food-relevant genome editing for developing dairy-compatible probiotic cultures.},
}

@article {pmid42806035,
year = {2026},
author = {Zhao, N and Olajide, JS and Guan, Z and Huang, J and Zou, X and Ou, F and Lin, J and Tao, T and Zhou, T and Gao, Z and Ren, Z and Shi, X and Huang, X and Wang, G},
title = {A dual A-to-Y and G-to-Y base editor and a four-base concurrent hypermutator in mammalian cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42806035},
issn = {2041-1723},
support = {82271909//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82300674//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32060040//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Cytidine Deaminase/metabolism/genetics ; Animals ; *Mutation ; CRISPR-Cas Systems ; Base Sequence ; DNA Glycosylases/genetics/metabolism ; Cytosine/metabolism ; Deoxyribonuclease I/metabolism/genetics ; HEK293 Cells ; AICDA (Activation-Induced Cytidine Deaminase) ; Mutagenesis ; Adenine/metabolism ; },
abstract = {Current dual base editors and hypermutators exhibit limited types of base conversion and constrained mutational diversity. To address this challenge, we engineer a compact dual base editor "A&GBE" by fusing a deaminase and glycosylase with nickase Cas9 (nCas9) to enable concurrent adenine and cytosine editing. Furthermore, we develop quadruple base mutating modules (QBMM) by fusing nickase or dead Cas9 with engineered thymine-DNA glycosylase (TDG), N-methylpurine DNA glycosylase (MPG) and TadA8e, together with MS2-MCP system recruited activation-induced cytidine deaminase (AID). This QBMM enables simultaneous mutation of all four types of nucleotide bases (A, T, G, C) within an approximately 100 base pair (bp) sequence surrounding the gRNA target site. This platform generates hyper diverse multi-nucleotide variants, establishing a powerful tool for accelerated protein evolution, functional genomics, and disease modeling.},
}

Humans
Cytidine Deaminase/metabolism/genetics
Animals
*Mutation
CRISPR-Cas Systems
Base Sequence
DNA Glycosylases/genetics/metabolism
Cytosine/metabolism
Deoxyribonuclease I/metabolism/genetics
HEK293 Cells
AICDA (Activation-Induced Cytidine Deaminase)
Mutagenesis
Adenine/metabolism

@article {pmid42806792,
year = {2026},
author = {Zhang, Q and Chang, S and Zhao, S and Zhang, W and Wang, R and Zhong, M and Liu, G and Li, X and Guo, A},
title = {In vivo CRISPR Screening Reveals LBR as a Regulator of CD8[+] T Cell Fate Decision.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {19},
pages = {e72318},
doi = {10.1096/fj.202602909R},
pmid = {42806792},
issn = {1530-6860},
support = {2023YFC2306500//Ministry of Science and Technology of China/ ; 82471755//National Natural Science Foundation of China/ ; 82502108//National Natural Science Foundation of China/ ; QYPY20230026//Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM/ ; 24YF2706400//Shanghai Science and Technology Innovation Action Plan/ ; },
mesh = {Animals ; *CD8-Positive T-Lymphocytes/immunology/metabolism/cytology ; *Cell Differentiation ; Mice ; *CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Knockout ; Cholesterol/metabolism ; Metabolic Reprogramming ; },
abstract = {CD8[+] T cells play an important role in anti-infection and anti-tumor immunity. Metabolic reprogramming has emerged as a critical regulator of T cell function, yet the roles of specific cholesterol-associated proteins during T cell fate trajectories remain unclear. Here, using an in vivo CRISPR screen in CD8[+] T cells during infection with a customized sgRNA library targeting key cholesterol metabolism-associated proteins, we successfully identified several candidate targets, including the LBR (Lamin B Receptor). Lbr knockout significantly reduced the proportion and absolute number of short-lived effector cells (SLECs), severely impairing effector responses, while concomitantly increasing the memory precursor population upon LCMV infection. Furthermore, transcriptomic analysis revealed that Lbr depletion markedly altered signaling pathways governing effector T cell differentiation. These findings reveal the role of LBR in modulating effector and memory T cell differentiation, suggesting it as a potential target for metabolic reprogramming to enhance T cell-based immunotherapies.},
}

Animals
*CD8-Positive T-Lymphocytes/immunology/metabolism/cytology
*Cell Differentiation
Mice
*CRISPR-Cas Systems
Mice, Inbred C57BL
*Clustered Regularly Interspaced Short Palindromic Repeats
Mice, Knockout
Cholesterol/metabolism
Metabolic Reprogramming

@article {pmid42807853,
year = {2026},
author = {Shandilya, UK and Liang, Y and Atalla, H and Mallard, B and Karrow, NA},
title = {Loss of IL10RA rewires epithelial immune responses to intracellular Staphylococcus aureus small colony variants in bovine mammary epithelial cells.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1891987},
pmid = {42807853},
issn = {2235-2988},
mesh = {Animals ; Cattle ; *Epithelial Cells/immunology/microbiology ; *Staphylococcus aureus/immunology/growth & development ; Female ; *Mammary Glands, Animal/immunology/microbiology/cytology ; *Staphylococcal Infections/immunology/veterinary/microbiology ; Mastitis, Bovine/microbiology/immunology ; *Interleukin-10 Receptor alpha Subunit/genetics/metabolism ; Gene Expression Profiling ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Staphylococcus aureus small colony variants (SCVs) represent a persistence-adapted phenotype linked to chronic and recurrent bovine mastitis. Interleukin-10 receptor ⍺ (IL10RA) is a central regulator of anti-inflammatory signalling in the mammary gland, but its role in shaping transcriptional responses to intracellular staphylococcal challenge has not been characterized.

METHODS: CRISPR/Cas9-generated IL10RA knockout (KO) MAC-T bovine mammary epithelial cells (MECs), alongside wild-type (WT) controls, were infected with either S. aureus SCV strain Heba3231 or its isogenic parental strain (PS). Transcriptional responses were profiled by RNA sequencing and compared across genotype and infection conditions.

RESULTS: WT cells mounted markedly divergent responses to PS (8 DEGs) versus SCV (461 DEGs) infection, with no overlap between the two gene sets, indicating strain-specific engagement of distinct molecular programmes. SCV infection of WT cells up-regulated lipid and sterol biosynthetic pathways while suppressing epithelial barrier integrity genes (S100A8, S100A9, TGM3, KRTDAP). IL10RA disruption substantially amplified transcriptional dysregulation: IL10RA-KO cells infected with PS yielded 619 DEGs (a 77.4-fold increase over WT-PS), while IL10RA-KO cells infected with SCV yielded 1,297 DEGs (a 2.8-fold increase over WT-SCV). Approximately 32 to 36% of IL10RA-regulated genes formed a shared core across infection conditions. Loss of IL10RA derepressed pro-inflammatory cytokine pathways (TNF, IL-17), enhanced pro-apoptotic programmes during SCV infection, and suppressed phagosomal maturation and lipid metabolic gene programmes. Candidate IL10RA-responsive genes, including CD79B, WFDC2, AMN and LY6E, were identified as targets for further validation.

DISCUSSION: These findings establish IL10RA as a broad transcriptional homeostasis regulator in bovine MECs during staphylococcal infection, with disproportionate effects on inflammatory and barrier-related gene programmes under SCV challenge. The identified candidate genes warrant validation in primary bovine mammary cells, milk somatic cells, or clinical mastitis samples to confirm their relevance to chronic and recurrent disease pathogenesis.},
}

Animals
Cattle
*Epithelial Cells/immunology/microbiology
*Staphylococcus aureus/immunology/growth & development
Female
*Mammary Glands, Animal/immunology/microbiology/cytology
*Staphylococcal Infections/immunology/veterinary/microbiology
Mastitis, Bovine/microbiology/immunology
*Interleukin-10 Receptor alpha Subunit/genetics/metabolism
Gene Expression Profiling
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid42809019,
year = {2026},
author = {Monfort, M and Brugière, N and Calbry, J and Lacou, L and Roeber, F and Nogué, F},
title = {Targeted allelic diversification of flowering genes via CRISPR-Cas enables the development of multiple early-flowering soybean lines.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {10},
pages = {},
pmid = {42809019},
issn = {1432-2242},
support = {ANR-22-PESV-0002//Agence Nationale de la Recherche/ ; },
mesh = {*Glycine max/genetics/growth & development/physiology ; *Flowers/genetics/growth & development/physiology ; *CRISPR-Cas Systems ; Alleles ; *Genes, Plant ; Genetic Variation ; Phenotype ; Photoperiod ; *Plant Breeding ; Plants, Genetically Modified ; },
abstract = {CRISPR-Cas multiplex editing generated a broad range of early-flowering soybean lines, advancing flowering by up to four weeks under controlled long-day conditions and by approximately one to two weeks in a field trial. Soybean is a promising crop for European agriculture, but its cultivation area remains smaller than its potential. A major limitation to broader adoption in Europe is the restricted number of elite lines adapted to local environmental conditions, particularly the photoperiod encountered at higher latitude. At these latitudes, soybean must flower early to complete its growth cycle before the onset of harsh autumn conditions. In this project, we employed a CRISPR-Cas multiplex knockout strategy, to generate allelic diversity in the gene network controlling flowering time, with the aim of developing earlier flowering soybeans lines better suited to European high latitudes. This approach successfully produced earlier flowering varieties, with flowering advances of up to four weeks compared to the controls. Starting from maturity group (MG) I and MG III lines, we developed, within two generations, gene-edited early flowering soybean lines with estimated maturity ranges between MG 000 and MG 0. Greenhouse observations were supported by a field trial, in an MG II environment, where edited lines flowered approximately one to two weeks earlier than their respective parental controls. These results demonstrate that multiplex editing of flowering-time genes can rapidly generate targeted allelic diversity and a broad spectrum of flowering phenotypes within elite soybean backgrounds. These results provide valuable breeding material and a genotype/phenotype resource for subsequent evaluation of flowering-time adaptation across environments.},
}

*Glycine max/genetics/growth & development/physiology
*Flowers/genetics/growth & development/physiology
*CRISPR-Cas Systems
Alleles
*Genes, Plant
Genetic Variation
Phenotype
Photoperiod
*Plant Breeding
Plants, Genetically Modified

@article {pmid42809066,
year = {2026},
author = {Ch, AA and Nazik, N and Shahid, M and Saleem, M and Nadeem, A and Saeed, F and Rao, AQ and Rashid, I and Bakhsh, A},
title = {Harnessing next-generation molecular technologies for precision control of Tuta absoluta and Helicoverpa armigera in tomato.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42809066},
issn = {1438-7948},
mesh = {Animals ; *Solanum lycopersicum/genetics/parasitology ; *Helicoverpa armigera/genetics/pathogenicity ; *Moths/genetics/pathogenicity ; RNA Interference ; *Pest Control, Biological/methods ; Plants, Genetically Modified/genetics/parasitology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Tomato (Solanum lycopersicum) is one of the most significant vegetable crops cultivated around the world. Among biotic stresses, production of tomato is severely affected by insect pests mainly by tomato fruit borer (Helicoverpa armigera) and tomato leaf miner (Tuta absoluta). Traditionally to manage the destruction caused by insect pests, farmers mostly used chemical insecticides and by different cultural practices. Use of chemical pesticides is of great concern as it is harmful for the environment and other living beings and with the passage of time, insects develop resistance against pesticides. Latest progress in molecular biology enables scientists to develop next generation pest management approaches that are more precise, environment friendly and sustainable. This review describes nascent molecular approaches to manage tomato pests, like transgenic expression of insecticidal proteins such as of Bacillus thuringiensis (Bt) toxins, RNAi (RNA interference) strategies including HIGS and SIGS, genome editing by CRISPR-Cas, protease inhibitors and male sterility. These strategies not only enhance resistance of crop against pests but also are safe for environment and other organisms. This review also addresses the combination of different molecular approaches and pyramiding of genes involve in delaying resistance. Overall, these advanced molecular approaches providing sustainable resistance against insect pests have been discussed.},
}

Animals
*Solanum lycopersicum/genetics/parasitology
*Helicoverpa armigera/genetics/pathogenicity
*Moths/genetics/pathogenicity
RNA Interference
*Pest Control, Biological/methods
Plants, Genetically Modified/genetics/parasitology
CRISPR-Cas Systems
Gene Editing

@article {pmid42811153,
year = {2026},
author = {Jeong, CS},
title = {AcrSeek: Metric Learning with Hybrid Negative Mining for Anti-CRISPR Protein Detection under Extreme Class Imbalance.},
journal = {Bioinformatics (Oxford, England)},
volume = {},
number = {},
pages = {},
doi = {10.1093/bioinformatics/btag729},
pmid = {42811153},
issn = {1367-4811},
abstract = {MOTIVATION: Anti-CRISPR (Acr) proteins inhibit CRISPR-Cas immunity and are key targets for precise control of CRISPR-based genome editing and phage-host coevolution research. Their experimental identification is costly and low-throughput, so computational predictors are used to prioritise candidates for testing. In this setting validated positives number in the hundreds while phage-derived putative negatives reach tens of thousands, producing extreme class imbalance. Yet existing classification-based predictors train and evaluate on 1:1 balanced datasets and allow high-similarity sequences between training and test, overestimating real-world performance.

RESULTS: We present AcrSeek, a metric-learning framework that trains a projection head over a frozen protein language model (PLM) encoder with hybrid negative mining (offline global plus online semi-hard) under a joint triplet-focal objective. Under nested 5-fold cross-validation with leakage controlled by 40% identity clustering, AcrSeek improved AUPRC on the imbalanced (1:383) test set from 0.017 to 0.355 over AcrNET (matched PLM backbone), reaching 0.508 with ESM-2 3B. AcrSeek therefore remains structurally robust under extreme class imbalance and may also serve other protein-function detection problems with scarce positives.

Source code at https://github.com/jeongchans/acrseek, archived at Zenodo (DOI: 10.5281/zenodo.21946803); data and model checkpoints at Zenodo (DOI: 10.5281/zenodo.20115442).

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.},
}

@article {pmid42814316,
year = {2026},
author = {Lu, D and Gong, X and Guo, X and Cai, Q and Li, W and Chen, Y and Li, D and Zeng, F},
title = {Precision base-editing of the cryptic 3' acceptor site to correct the RNA splicing defect of β[654]-thalassemia.},
journal = {Molecular biomedicine},
volume = {7},
number = {1},
pages = {},
pmid = {42814316},
issn = {2662-8651},
support = {2024YFC2707002//the National Key Research and Development Program of China/ ; 2024YFC2707001//the National Key Research and Development Program of China/ ; 82000186//the National Natural Science Foundation of China/ ; 82271890//the National Natural Science Foundation of China/ ; 32570951//the National Natural Science Foundation of China/ ; 31871484//the National Natural Science Foundation of China/ ; U23A20498//the National Natural Science Foundation of China/ ; 22ZR1451600//the Natural Science Foundation of Shanghai/ ; 0092/2022/A2//the Macau Science and Technology Development fund (FDCT)/ ; 003/2022/ALC//the Macau Science and Technology Development fund (FDCT)/ ; },
mesh = {Animals ; *beta-Thalassemia/genetics/therapy ; *RNA Splicing/genetics ; *RNA Splice Sites/genetics ; Mice ; *beta-Globins/genetics ; *Gene Editing/methods ; Mutation ; Humans ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Female ; },
abstract = {β-Thalassemia is one of the most common inherited disorders worldwide and is caused by mutations affecting β-globin production. β[654] mutation (IVS2-654, C > T) is one of the most frequently occurring β-thalassemia alleles in Han Chinese population, which activates a cryptic 3' splice site and leads to aberrant RNA splicing. Previous studies demonstrated that direct base editing of the IVS2-654 mutation is challenging because suitable single-guide RNAs (sgRNAs) cannot effectively target this site. Here we investigated an alternative therapeutic strategy by targeting the mutation-activated cryptic 3' splice acceptor site, rather than the disease-causing mutation itself. We first introduced base substitutions into the cryptic splice acceptor site in a β[654]-thalassemia mouse model using CRISPR-Cas9-mediated homology-directed repair, which restored normal RNA splicing and validated the therapeutic rationale of this approach. We then generated base-edited β[654] mice by microinjecting Td-CBEmax mRNA together with sgRNAs targeting the cryptic splice acceptor site into one-cell embryos. Base editing was successfully achieved in 78% of live-born β[654] mice, of which 86% produced correctly spliced β-globin transcripts. Restoration of normal RNA splicing was accompanied by marked improvement of hematological parameters and tissue pathology in most base-edited founder mice and their offspring compared with non-edited β[654] mice. Together, these findings demonstrate that mutation-activated cryptic splice sites are therapeutically actionable targets for precision base editing and provide proof of concept for a precise and effective strategy to correct aberrant RNA splicing in β[654]-thalassemia.},
}

Animals
*beta-Thalassemia/genetics/therapy
*RNA Splicing/genetics
*RNA Splice Sites/genetics
Mice
*beta-Globins/genetics
*Gene Editing/methods
Mutation
Humans
Disease Models, Animal
CRISPR-Cas Systems/genetics
Female

@article {pmid42814784,
year = {2026},
author = {Wu, J and Shi, W and Luo, S and Armlich, LM and Wu, Y and Liu, Q and Chen, X and Eliseev, R and Kretzler, NK and Halavath, SK and Guan, P and Nick, P},
title = {VvDLK negatively regulates cold tolerance by interacting with Vv14-3-3A in grapevine.},
journal = {Plant physiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/plphys/kiag728},
pmid = {42814784},
issn = {1532-2548},
abstract = {Sudden cold snaps due to blurred seasonality are a rising challenge to agriculture in times of climate change. Swift activation of cold signaling is crucial for resilience. Dual-localization kinesins (DLK) convey the cold signal from the plasma membrane to the nucleus, where this protein modulates cold-dependent gene expression. Using the grapevine homologue as paradigm, we investigated the functional context of VvDLK. We combined live-cell and immunofluorescence imaging with pharmacological interference, physiological analysis and protein-interaction assays upon stable expression in grapevine cells and tomato, or CRISPR-Cas mediated inactivation of the tomato homologue. Overexpression of VvDLK was associated with earlier and more pronounced cold-induced elimination of microtubules in both, tomato plants as heterologous, and grapevine cells as homologous host, followed by reduced cold tolerance, evident from cellular, physiological, and molecular readouts. Conversely, inactivation of the tomato VvDLK homologue SlDLK improved cold tolerance. VvDLK interacted specifically with Vv14-3-3A, and a 14-3-3 inhibitor attenuated cold-induced microtubule loss. Overexpression of Vv14-3-3A also increased cold susceptibility. In addition, VvDLK and Vv14-3-3A both interacted with VvCBF4, while VvDLK reduced VvCBF4 protein accumulation and altered its intranuclear distribution under cold stress. Together, these findings suggest that VvDLK is associated with cold-induced microtubule elimination and attenuates cold tolerance through a signaling module involving Vv14-3-3A and VvCBF4.},
}

@article {pmid42814837,
year = {2026},
author = {Moescheid, MF and Wisitpongpun, P and Gross, V and Mann, VH and Quack, T and Rognon, A and Grunau, C and Ittiprasert, W and Brindley, PJ and Grevelding, CG},
title = {Toward transgenic multicellular parasites: CRISPR-Cas12a germline editing of Schistosoma mansoni eggs as key to success.},
journal = {Science advances},
volume = {12},
number = {40},
pages = {eaeh0455},
pmid = {42814837},
issn = {2375-2548},
support = {/WT_/Wellcome Trust/United Kingdom ; HHSN272201700014C/AI/NIAID NIH HHS/United States ; R01 CA164719/CA/NCI NIH HHS/United States ; S10 RR025565/RR/NCRR NIH HHS/United States ; },
mesh = {Animals ; *Schistosoma mansoni/genetics ; *CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; *Gene Editing/methods ; Cricetinae ; Green Fluorescent Proteins/genetics ; Ovum/metabolism ; Germ Cells/metabolism ; },
abstract = {Lack of transgenesis methods impedes functional studies in platyhelminths. We present a substantially improved ribonucleic protein complex (RNP)-based genome editing approach for Schistosoma mansoni comparing Cas9 and Cas12a, the latter attained more efficient homology-directed insertion of a 5'-C6-PEG10-modified double-stranded donor template bearing 50-nucleotide microhomology arms into a genome safe-harbor site (GSH1). Electroporation of eggs recovered from host livers resulted in enhanced green fluorescent protein (EGFP) reporter activity, driven by an S. mansoni ubiquitin promoter and terminator. Hamster infection with transgenic cercariae, the infectious stage of this parasitic platyhelminth, recovered from miracidial infection of snails, or from sporocysts generated in vitro from transgenic miracidia and implantation in snails, produced EGFP-positive adult worms. Sequencing of the edited GSH1 and analyses of filial generations demonstrated chromosomal integration and the stable transformation of S. mansoni. Together, this approach advances schistosome transgenesis and may be pioneering for other platyhelminths, which lack functional genomics and transformation methods.},
}

Animals
*Schistosoma mansoni/genetics
*CRISPR-Cas Systems/genetics
Animals, Genetically Modified
*Gene Editing/methods
Cricetinae
Green Fluorescent Proteins/genetics
Ovum/metabolism
Germ Cells/metabolism

@article {pmid42031104,
year = {2026},
author = {Ren, Q and Gao, X and Shi, T and Cao, J},
title = {Fusing T7 exonuclease with the G-to-T base editor enhances the editing purity in rice.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {10},
pages = {1998-2007},
doi = {10.1016/j.jgg.2026.04.010},
pmid = {42031104},
issn = {1673-8527},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; INDEL Mutation ; *Exonucleases/genetics/metabolism ; },
abstract = {The G-to-T base editor (GTBE) is a genome editing tool that does not rely on deaminases. However, its application has been limited by low G-to-T editing efficiency and high insertion and deletion (InDel) byproduct rates. In this study, we develop a series of editors (GTBE1-GTBE7) using three strategies: modification of the Cas9 nickase (nCas9) variant, fusion with a transcriptional activation domain, and fusion with a 5'-3' exonuclease targeting nicked DNA. Both protoplast and stable rice transformant analyses reveal that the GTBE7 editor, which incorporates T7 exonuclease, significantly improves G-to-T editing purity compared with the previously reported GTBE1 editor. In stable transformants, the InDel efficiency of GTBE7 is reduced by 14%-27.7% compared with GTBE1. To further broaden the editing range of GTBE7, we develop the GTBE7_nSpRY editor, enabling G-to-T base editing at non-NGG protospacer adjacent motif (PAM) sequences. Using GTBE7_nSpRY, we create herbicide-resistant rice and identify the G628A mutation site, generating imazethapyr-resistant rice plants through G-to-C byproduct edits. In summary, the GTBE7 and GTBE7_nSpRY editors developed in this study enhance G-to-T editing purity, reduce InDel byproduct efficiency, and offer potential for expanding the application of G-to-T base editing in plants.},
}

*Oryza/genetics
*Gene Editing/methods
CRISPR-Cas Systems/genetics
INDEL Mutation
*Exonucleases/genetics/metabolism

@article {pmid42586203,
year = {2026},
author = {Jiang, W and Sui, X and Dong, D and Jiang, L and Zheng, X and Wang, H and Li, B and Hou, L and Liang, Y and Jia, M and Jiang, S and Teng, H and Wang, W},
title = {Genome-scale CRISPR screening uncovers SRSF6 as a target to sensitize hepatocellular carcinoma to radiotherapy.},
journal = {JHEP reports : innovation in hepatology},
volume = {8},
number = {10},
pages = {101978},
pmid = {42586203},
issn = {2589-5559},
mesh = {Humans ; *Carcinoma, Hepatocellular/radiotherapy/genetics ; *Liver Neoplasms/radiotherapy/genetics ; Animals ; Mice ; *Radiation Tolerance/genetics/drug effects ; Cell Line, Tumor ; *Serine-Arginine Splicing Factors/genetics/metabolism/antagonists & inhibitors ; Hep G2 Cells ; Mice, Inbred BALB C ; Mice, Nude ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND & AIMS: Radiotherapy confers clinical benefits to patients with hepatocellular carcinoma (HCC) across all stages, yet its clinical efficacy is limited by radioresistance. This study aimed to identify key regulators of HCC radiosensitivity through genome-wide functional screening.

METHODS: A genome-wide CRISPR-Cas9 screen in Huh7 cells identified radiosensitivity regulators, with SRSF6 validated by siRNA knockdown and γ-H2AX assessment. Stable shRNA-mediated SRSF6 knockdown was established in Huh7 and HepG2 cells, followed by clonogenic, EdU incorporation, apoptosis, micronucleus, and comet assays. Mechanistically, RNA-seq, Western blotting, mRNA stability assays, RIP-qPCR, and RAD51 overexpression rescue assays were performed. The therapeutic potential of the SRSF6 inhibitor indacaterol was evaluated using MTS assays, HCC xenograft mouse models (BALB/c-nu/nu, n = 28), and HCC patient-derived organoids (PDOs) (n = 3). In addition, SRSF6 expression and its correlation with patient survival were analyzed using data from The Cancer Genome Atlas and a tissue microarray (n = 14 HCC and 14 paired adjacent non-tumorous liver samples).

RESULTS: We identified the RNA-binding protein SRSF6 as a driver of HCC radioresistance. SRSF6 depletion enhanced the radiosensitivity of HCC cells (p <0.05-0.0001) by post-transcriptionally destabilizing the mRNAs of critical DNA repair genes (p <0.05-0.0001), thereby impairing radiation-induced DNA damage repair. The radiosensitizing effect of SRSF6 depletion was partially abrogated by ectopic overexpression of the core DNA repair protein RAD51 (p <0.05-0.001). Indacaterol exhibited cytotoxic effects on HCC cells (p <0.05-0.0001) and enhanced the antitumor efficacy of radiation in vivo (p <0.05-0.0001), as further validated across multiple HCC patient-derived organoids (p <0.05-0.0001).

CONCLUSIONS: SRSF6 is a key regulator of HCC radioresistance through its post-transcriptional control of DNA repair capacity, and represents a novel therapeutic target to sensitize HCC to radiotherapy.

IMPACT AND IMPLICATIONS: In this study, we performed a genome-wide CRISPR-Cas9 knockout library screen to dissect the molecular determinants governing HCC radiosensitivity, and identified RNA-binding protein SRSF6 as a driver of HCC radioresistance. We demonstrate that SRSF6 depletion disrupts the post-transcriptional stability of key DNA repair gene mRNAs and enhances HCC radiosensitivity. These findings are important for radiation oncologists and translational researchers, as they identify SRSF6-dependent RNA regulation as a critical determinant of radiotherapy response in HCC. Practically, we show that the clinically approved bronchodilator indacaterol suppresses SRSF6 function and enhances the antitumor efficacy of radiotherapy, offering a readily repurposable pharmacological strategy to overcome radioresistance. These implications are based on preclinical evidence across multiple models; however, future clinical trials are needed to validate the safety and efficacy of indacaterol-based radiosensitization in patients with HCC.},
}

Humans
*Carcinoma, Hepatocellular/radiotherapy/genetics
*Liver Neoplasms/radiotherapy/genetics
Animals
Mice
*Radiation Tolerance/genetics/drug effects
Cell Line, Tumor
*Serine-Arginine Splicing Factors/genetics/metabolism/antagonists & inhibitors
Hep G2 Cells
Mice, Inbred BALB C
Mice, Nude
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid42799621,
year = {2026},
author = {Sun, J and Tan, P and Shao, Y and Lu, Z},
title = {CRISPR/Cas9-Mediated HKDC1 Depletion to Assess Ferroptosis and Lenvatinib Responses in Hepatocellular Carcinoma.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/73461},
pmid = {42799621},
issn = {1940-087X},
mesh = {Humans ; *Quinolines/pharmacology ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism/enzymology ; *Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism/enzymology ; *Phenylurea Compounds/pharmacology ; *Ferroptosis/drug effects/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; },
abstract = {Hexokinase domain-containing protein 1 (HKDC1) has been implicated in hepatocellular carcinoma (HCC) progression, but its relationship to regulated cell death remains incompletely defined. This article presents an in vitro workflow that combines public cohort analysis, paired clinical tissues, CRISPR/Cas9-mediated HKDC1 depletion, cell growth assays, lenvatinib treatment, pharmacological rescue, and biochemical measurements of glutathione (GSH) and malondialdehyde (MDA). HKDC1 expression was higher in HCC than in non-tumor liver tissue and was associated with poorer overall and disease-free survival in public datasets. HKDC1 depletion reduced growth and colony formation in Hep3B and SNU449 cells. HKDC1 depletion also increased the response to lenvatinib and ML-162. Independent targeting with sg-HKDC1-2 reproduced the ML-162-associated viability phenotype, and ferrostatin-1 restored viability after ML-162 exposure in SNU449 cells. These results support an association between HKDC1 depletion and increased susceptibility to regulated cell-death stress in HCC cells. Because direct lipid-reactive oxygen species, GPX4/SLC7A11 activity, glucose flux, and in vivo efficacy were not measured, the downstream mechanism and therapeutic relevance require further validation.},
}

Humans
*Quinolines/pharmacology
*Liver Neoplasms/genetics/drug therapy/pathology/metabolism/enzymology
*Carcinoma, Hepatocellular/genetics/drug therapy/pathology/metabolism/enzymology
*Phenylurea Compounds/pharmacology
*Ferroptosis/drug effects/genetics
Cell Line, Tumor
*CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology

@article {pmid42799886,
year = {2026},
author = {Lagner, J and Paulson, A and Schulden, T and Adhikari, B and Koob, J and Vakulskas, C and Qi, Y},
title = {CRISPR-Cas13-based amplification-free detection of three quarantine-significant sugarcane viruses.},
journal = {Plant cell reports},
volume = {45},
number = {10},
pages = {},
pmid = {42799886},
issn = {1432-203X},
support = {AP23PPQS&T00C106//Animal and Plant Health Inspection Service/ ; IOS- 2132693//Directorate for Biological Sciences/ ; },
mesh = {*Saccharum/virology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; *Potyviridae/genetics/isolation & purification ; *Plant Viruses/genetics/isolation & purification ; RNA, Viral/genetics ; *Potyvirus/genetics/isolation & purification ; Luteoviridae ; },
abstract = {Crop losses from viral pathogens cost billions of dollars annually, and sugarcane is particularly vulnerable. Sugarcane is the most cultivated crop in the world by volume; therefore, significant yield losses result in large dollar losses for farmers and trade economies. In some cases, viruses contribute to more than half of the yield reduction. Three quarantine-significant pathogens-Sugarcane mosaic virus (ScMV), Sugarcane streak mosaic virus (ScSMV), and Sugarcane yellow leaf virus (ScYLV)- pose major threats to global sugarcane production because of their rapid spread, asymptomatic infections, and delayed symptom expression. Effective management of these pathogens requires robust, rapid, and accurate detection methods that can distinguish among these viruses, even at low titers. Here, we report the development of an amplification-free CRISPR-Cas13-based assay with a limit of detection (LOD) of ~1.5 pM. The assay allows for simultaneous screening of all three viruses directly from bulk RNA extracts of plant material. Our approach leverages LwaCas13a specificity to detect and differentiate viral targets in a single assay. Compared with conventional multi-step assays, this strategy significantly reduces time and contamination risk. Validation on naturally infected material demonstrated its sensitivity and reliability. This platform provides a powerful tool for early and accurate diagnosis of ScMV, ScSMV, and ScYLV, with strong potential for integration into sugarcane virus surveillance and management programs.},
}

*Saccharum/virology
*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
*Potyviridae/genetics/isolation & purification
*Plant Viruses/genetics/isolation & purification
RNA, Viral/genetics
*Potyvirus/genetics/isolation & purification
Luteoviridae

@article {pmid42801307,
year = {2026},
author = {Pasari, N and Bhandhari, N and Shruti, and Kumar, R and Yazdani, SS and Sinharoy, S},
title = {CRISPR-Cas9 Genome Editing in Mesorhizobium ciceri Through nodC Disruption for Chickpea Symbiosis Studies.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {235},
pages = {},
doi = {10.3791/71513},
pmid = {42801307},
issn = {1940-087X},
mesh = {*Cicer/microbiology/physiology ; *Mesorhizobium/genetics/physiology ; Symbiosis/genetics ; *CRISPR-Cas Systems ; *N-Acetylglucosaminyltransferases/genetics ; *Gene Editing/methods ; },
abstract = {Mesorhizobium ciceri, a nitrogen-fixing symbiont of chickpea (Cicer arietinum), remains genetically challenging to manipulate using conventional homologous recombination approaches, which are labor-intensive and often leave undesirable selection markers. In this protocol, we describe a streamlined genome-editing strategy using a broad-host-range Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) system adapted for M. ciceri. We demonstrate the efficacy of this method by targeting nodC, which encodes the N-acetylglucosaminyltransferase required for chitin backbone synthesis in Nod factors, the primary signaling molecules involved in symbiotic molecular communication. This protocol details the systematic design of single-guide RNAs (sgRNAs) and the construction of a homology-directed repair (HDR) template. The HDR template was designed to facilitate site-specific integration of a green fluorescent protein (GFP) reporter flanking the nodC cleavage site. Following delivery of the Cas9/sgRNA/HDR construct through biparental mating, putative mutants were identified using a fluorescence-based screening approach. Successful disruption of the 1.3 kb nodC locus within the nodulation (nod) cassette was initially screened by visualization of GFP expression in mutant colonies using fluorescence microscopy. The disruption was further validated by restriction digestion, amplification of the integrated cassette from genomic DNA, and Sanger sequencing. GFP expression was additionally quantified by reverse transcription quantitative polymerase chain reaction. To validate the functional impact of the mutation, chickpea infection assays were performed, demonstrating impaired nodulation in plants inoculated with ΔnodC M. ciceri compared with the wild-type strain. Overall, this protocol provides an efficient and reproducible framework for precise gene disruption and functional genomics studies in Mesorhizobium.},
}

*Cicer/microbiology/physiology
*Mesorhizobium/genetics/physiology
Symbiosis/genetics
*CRISPR-Cas Systems
*N-Acetylglucosaminyltransferases/genetics
*Gene Editing/methods

@article {pmid42801364,
year = {2026},
author = {Khan, S and Ilyas, N and Nawaz, A and Nawaz, H and Khan, MI and Bhatti, MT and Hussain, U and Khan, S},
title = {Anti-phage defense systems in bacteria: molecular mechanisms and their role in shaping phage therapy strategies.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42801364},
issn = {1573-4978},
support = {No. 20-16428/NRPU/R&D/HEC/2021//Higher Education Commision, Pakistan/ ; },
mesh = {*Bacteriophages/genetics/immunology ; *Bacteria/virology/genetics/immunology ; *Phage Therapy/methods ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; },
abstract = {Bacteria and phages are engaged in a persistent evolutionary struggle. To survive constant phage predation, bacteria have evolved a highly diverse and multi-layered immune arsenal that determines the success of therapeutic phage infection. Bacterial defenses include receptor blockade, DNA restriction systems, CRISPR-Cas adaptive immunity, and secondary messenger signaling systems that induce effector-mediated cell death. Recent mechanistic advances have elucidated systems such as Thoeris (gcADPR-activated SIR2 effectors depleting NAD[+]), CBASS (cyclic nucleotide-activated effectors disrupting cell integrity), and toxin-antitoxin systems (e.g., ShosTA disrupting purine metabolism). These defenses directly impact phage therapy outcomes. However, phages have evolved sophisticated countermeasures, including RNA-based anti-CRISPRs and enolase hijacking, while engineered phages carrying synthetic anti-defense proteins are being developed to overcome bacterial immunity. The present review integrates defense system classification, phage counter-defense evolution, and their associations with phage therapy outcomes within a unified framework for phage selection and engineering. This review provides a scientific basis for defense-informed phage selection, rational phage engineering, and the design of future clinical trials against multidrug-resistant infections.},
}

*Bacteriophages/genetics/immunology
*Bacteria/virology/genetics/immunology
*Phage Therapy/methods
CRISPR-Cas Systems
Host-Pathogen Interactions

@article {pmid42468175,
year = {2027},
author = {Xu, Z and Gu, Z and Ma, Z and Hu, B and Ye, BC and Xu, H},
title = {CRISPR-responsive hybrid MOF assembling assay for rapid electrochemical detection of domoic acid.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130297},
doi = {10.1016/j.talanta.2026.130297},
pmid = {42468175},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Kainic Acid/analogs & derivatives/analysis ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; Saxitoxin/analysis ; },
abstract = {Domoic acid (DA), a potent neurotoxin, poses a serious threat to food safety and public health. Traditional detection methods often struggle to balance sensitivity with practical applicability. To address this, we developed an electrochemical biosensor based on a CRISPR-responsive hybrid metal-organic framework (MOF) assembling assay. This platform employs a novel negative feedback mechanism where the DA aptamer itself serves as the activator for CRISPR/Cas12a. Target binding inhibits Cas12a's trans-cleavage activity, thereby protecting the signal reporter from cleavage and enabling significant signal amplification. The system integrates dual signal amplification by using rolling circle amplification (RCA)-generated DNA nanowires as scaffolds on magnetic beads and UiO-66 MOFs loaded with abundant methylene blue (MB) molecules as efficient signal tags. This synergistic strategy effectively enhances the signal response while minimizing background noise. When coupled with portable paper-based electrodes for differential pulse voltammetry (DPV) readout, the biosensor achieved highly sensitive DA detection with a low limit of detection. Furthermore, it exhibited robust universality and specificity by successfully detecting saxitoxin (STX) in complex matrices through simple aptamer substitution, demonstrating its modular design advantage. This work not only provides a reliable and practical solution for on-site DA monitoring but also establishes a versatile and powerful biosensing platform with significant potential for point-of-care testing of a broad spectrum of hazardous substances.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Kainic Acid/analogs & derivatives/analysis
*Metal-Organic Frameworks/chemistry
*CRISPR-Cas Systems
Limit of Detection
Aptamers, Nucleotide/chemistry
Saxitoxin/analysis

@article {pmid42470851,
year = {2027},
author = {Xu, K and Wang, S and Zhang, K and Zhang, A and Zhang, Y and Yu, S and Tang, Y},
title = {CRISPR/Cas12a-coupled organic electrochemical transistors for highly sensitive detection of methylated DNA.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130324},
doi = {10.1016/j.talanta.2026.130324},
pmid = {42470851},
issn = {1873-3573},
mesh = {*DNA Methylation ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods/instrumentation ; *Biosensing Techniques/methods ; Humans ; *Transistors, Electronic ; *DNA/analysis/genetics ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methylation is an important epigenetic biomarker for early disease screening and prognosis evaluation, but its reliable detection remains challenging because methylated DNA is often present at low abundance in complex biological backgrounds. Here, we report a methylation-sensitive bioelectronic sensing platform that integrates AciI-assisted target discrimination, CRISPR/Cas12a-mediated trans-cleavage, and vertical organic electrochemical transistors (vOECTs) amplification for highly sensitive methylated DNA detection. In this strategy, unmethylated DNA is selectively digested by AciI, while intact methylated DNA activates the crRNA-guided Cas12a system, triggering collateral cleavage of ssDNA reporters immobilized on the Au gate electrode. The resulting interfacial changes are efficiently amplified by the vOECTs through coupled electric-double-layer gating. The platform achieved quantitative methylated DNA detection from 100 fM to 100 pM with a sensitivity of 267.6 μA/dec and a detection limit of 100 fM. The sensor also exhibited good operational stability, reproducibility, and reliable recovery performance in artificial serum samples. This work demonstrates the potential of CRISPR/vOECTs bioelectronics for sensitive epigenetic analysis and presents a promising proof-of-concept for future non-invasive screening strategies.},
}

*DNA Methylation
*CRISPR-Cas Systems
*Electrochemical Techniques/methods/instrumentation
*Biosensing Techniques/methods
Humans
*Transistors, Electronic
*DNA/analysis/genetics
Limit of Detection
Reproducibility of Results
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42520632,
year = {2027},
author = {Zhao, C and Liu, X and Sun, R and Kong, F and Wang, J and Wang, D},
title = {Amplification-free detection of N6-Methyladenosine (m6A) based on droplet digital CRISPR-Cas13a.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130370},
doi = {10.1016/j.talanta.2026.130370},
pmid = {42520632},
issn = {1873-3573},
mesh = {*Adenosine/analogs & derivatives/analysis ; *CRISPR-Cas Systems ; RNA Methylation ; Humans ; RNA, Messenger/genetics ; *RNA/genetics ; },
abstract = {m6A (N6-methyladenosine), a prevalent RNA modification in eukaryotic mRNA involved in multiple biological processes, enables early cancer screening via its detection. m6A methylation detection is considered challenging, with most existing methods being limited by complex chemical transformations that incur high cost and procedural complexity. To overcome these challenges, a novel droplet-based digital CRISPR-Cas13a (Dd-Cas13a) approach was developed for the detection of m6A modifications in RNA. The method employs an m6A-specific antibody enrichment strategy to selectively enrich RNA molecules harboring m6A modifications, while RNA molecules lacking m6A remain unselected. Integration of the targeted recognition capability of the CRISPR/Cas13a system with droplet technology enables amplification-free detection of m6A-modified RNA at fM concentrations, with a significant improvement in sensitivity. The Dd-Cas13a detection platform enables visualization and quantitative analysis of methylation site proportions in cellular RNA, providing a novel approach for rapid and sensitive detection of RNA methylation, with significant potential for applications in early disease diagnosis and therapeutic efficacy assessment.},
}

*Adenosine/analogs & derivatives/analysis
*CRISPR-Cas Systems
RNA Methylation
Humans
RNA, Messenger/genetics
*RNA/genetics

@article {pmid42561795,
year = {2027},
author = {Zhu, Y and Luo, S and Luo, M and Liu, J and Lou, Y and Fan, R and Pan, J and Chen, H and Zhang, Y and Zhu, J and Zhang, Y and Zhang, X and Lin, C and Gao, Z},
title = {Photoactivated liquid-liquid phase separation-based cascade CRISPR/Cas12a electrochemical platform for sensitive small extracellular vesicles detection.},
journal = {Talanta},
volume = {312},
number = {Pt A},
pages = {130374},
doi = {10.1016/j.talanta.2026.130374},
pmid = {42561795},
issn = {1873-3573},
mesh = {Humans ; Phase Separation ; *Extracellular Vesicles/metabolism/chemistry ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *Stomach Neoplasms/diagnosis ; Limit of Detection ; Biomarkers, Tumor ; },
abstract = {Small extracellular vesicles (sEVs) have emerged as promising liquid biopsy biomarker for gastric cancer (GC) diagnosis. However, profiling sEVs surface proteins in complex clinical matrices remains challenging by limited sensitivity and high background interference. In this study, we developed an electrochemical platform based on the photoactivated liquid-liquid phase separation cascaded Cas12a amplification (PL-cCas12a) for ultrasensitive and specific detection of sEVs surface proteins. The rationally designed system enables precise spatiotemporal control over the reaction. Photoactivation ensures precise reaction initiation. The spatially confined microenvironment accelerate reaction efficiency by local substrates enrichment and suppresses non-specific trans-cleavage leakage via steric hindrance. The PL-cCas12a platform achieved the limit of detection (LOD) as low as 3.95 × 10[3] particles/μL with negligible background leakage. By integrating the aptamers targeting GC-associated sEV surface proteins, this platform accurately discriminated GC patients from healthy donors (AUC = 0.912). Therefore, this platform represents a promising tool for non-invasive early cancer diagnosis.},
}

Humans
Phase Separation
*Extracellular Vesicles/metabolism/chemistry
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*Stomach Neoplasms/diagnosis
Limit of Detection
Biomarkers, Tumor

@article {pmid42600293,
year = {2026},
author = {Zhang, Y and Wu, S and Chen, Y},
title = {Portable and sensitive detection of peanut allergen Ara h 1 via a CRISPR/Cas12a-integrated personal glucose meter using polydopamine-functionalized pipette tips.},
journal = {Food chemistry},
volume = {526},
number = {},
pages = {150780},
doi = {10.1016/j.foodchem.2026.150780},
pmid = {42600293},
issn = {1873-7072},
mesh = {*Biosensing Techniques/instrumentation/methods ; Polymers/chemistry ; *Arachis/chemistry/genetics/immunology ; *Plant Proteins/analysis ; Indoles/chemistry ; *Antigens, Plant/analysis ; *Glycoproteins/analysis ; CRISPR-Cas Systems ; Glucose/analysis ; Limit of Detection ; Membrane Proteins ; },
abstract = {A portable biosensor for the peanut allergen Ara h 1 was developed by integrating a polydopamine-functionalized pipette tip, CRISPR/Cas12a signal amplification, and a personal glucose meter. Ara h 1 binding induced partial displacement of a cDNA activator, which activated Cas12a trans-cleavage and released glucose oxidase from GOx-DNA conjugates. The released enzyme catalyzed glucose oxidation, producing a quantitative decrease in glucose concentration. Under optimized conditions, the assay showed a linear range of 50-1000 ng/mL and a solution-based limit of detection of 22.19 ng/mL. Post-extraction matrix-spike tests in commercial peanut-biscuit and peanut-beverage extracts yielded recoveries of 101.58-104.68% with relative standard deviations below 3.44%. These results demonstrate the analytical feasibility and preliminary matrix compatibility of the platform, although validation using pre-extraction fortified, incurred, and naturally contaminated foods is still required.},
}

*Biosensing Techniques/instrumentation/methods
Polymers/chemistry
*Arachis/chemistry/genetics/immunology
*Plant Proteins/analysis
Indoles/chemistry
*Antigens, Plant/analysis
*Glycoproteins/analysis
CRISPR-Cas Systems
Glucose/analysis
Limit of Detection
Membrane Proteins

@article {pmid42604703,
year = {2026},
author = {Du, J and Liu, L and Gao, J and Zhu, X and Kong, L and Cui, T and Cai, M and Li, X and Zhao, Y and Lu, X and Wang, N and Zhang, S},
title = {The spatiotemporal-specific regulation of cell wall-related proteins promotes the secretion of pigments in Antarctic fungi Geomyces sp. wnf-18c.},
journal = {Bioresource technology},
volume = {462},
number = {},
pages = {135614},
doi = {10.1016/j.biortech.2026.135614},
pmid = {42604703},
issn = {1873-2976},
mesh = {*Cell Wall/metabolism ; *Pigments, Biological/metabolism ; Antarctic Regions ; *Fungal Proteins/metabolism/genetics ; Gene Expression Regulation, Fungal ; CRISPR-Cas Systems/genetics ; Hypocreales ; },
abstract = {The polar environment harbors extremely rich and valuable natural product resources. In this study, a strain of Antarctic fungus, Geomyces sp. wnf-18c, in Antarctic soil was isolated in a laboratory. This fungus produces a purplish-red pigment whose hue closely resembles that of the high-end pigment carminic acid. However, approximately 50 % of the pigment remains within the fungal hyphae, restricting subsequent separation and extraction. In this study, the deletion of the key cell wall protein-encoding genes CHS3 and AGS1 enhanced pigment secretion. It also considerably inhibited the strain's growth, making it difficult to substantially increase the total pigment yield. To overcome these hurdles, the xylose-inducible system from Trichoderma reesei was heterologously expressed in Antarctic fungi, and the CRISPR-Cas9 system was placed under the control of this induction system. The exogenous addition of xylose as a signal to initiate genetic editing achieved the spatiotemporal regulation of the target gene-activating gene editing that restricts growth only after the completion of the strain's growth phase. The spatiotemporal regulation of key cell wall proteins significantly mitigated growth inhibition, greatly enhanced pigment secretion, and raised the total pigment yield. This discovery advances our understanding of the spatiotemporal specificity and dynamic regulation of microorganisms, offering novel strategies for exploiting polar microbial resources and reducing industrial production costs.},
}

*Cell Wall/metabolism
*Pigments, Biological/metabolism
Antarctic Regions
*Fungal Proteins/metabolism/genetics
Gene Expression Regulation, Fungal
CRISPR-Cas Systems/genetics
Hypocreales

@article {pmid42607467,
year = {2027},
author = {Zhou, Y and Zhang, H and Gao, H and Zhu, H and Liu, Y and Ma, H and Pan, H and Rong, S and Chang, D},
title = {A cascade colorimetric sensor based on Meso-UiO-66(Zr)@PtPdNPs nanozyme with triple enzyme activities and RPA-CRISPR/Cas12a for Staphylococcus aureus detection.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130451},
doi = {10.1016/j.talanta.2026.130451},
pmid = {42607467},
issn = {1873-3573},
mesh = {*Staphylococcus aureus/isolation & purification ; *Colorimetry/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Bacterial Proteins ; Metal-Organic Frameworks ; Endodeoxyribonucleases ; Phthalic Acids ; CRISPR-Associated Proteins ; },
abstract = {Staphylococcus aureus (S. aureus) is a common foodborne pathogen that can cause severe illnesses such as food poisoning and toxic shock syndrome, posing a significant threat to public health. Therefore, rapid and highly sensitive detection of S. aureus is crucial for ensuring food safety. In this study, a Meso-UiO-66(Zr)@PtPdNPs nanozyme with triple enzyme activities was synthesized and combined with the RPA-CRISPR/Cas12a system to construct a novel colorimetric platform for S. aureus detection. This platform utilizes the oxidase and peroxidase activities of Meso-UiO-66(Zr)@PtPdNPs to achieve self-driven catalytic cascade signal amplification without the need for external H2O2. During the assay, RPA first amplifies the target DNA to activate the trans-cleavage function of CRISPR/Cas12a. The activated CRISPR/Cas12a then degrades the magnetic bead probe (SMBs-S2), preventing it from coupling with the nanozyme probe (Meso-UiO-66(Zr)@PtPdNPs-S1). After magnetic separation, the nanozyme in the precipitate efficiently catalyzes the color development of TMB through the OXD-POD cascade effect. The resulting color intensity reflects the S. aureus levels. This biosensor shows excellent sensitivity and specificity by integrating the specific recognition and cleavage capabilities of the CRISPR system with the robust catalytic performance of the nanozyme. It displays a linear range of 1.5 × 10[1]-1.5 × 10[8] CFU/mL, alongside a 2.6 CFU/mL detection limit. This study provides a novel strategy for the detection of S. aureus in food.},
}

*Staphylococcus aureus/isolation & purification
*Colorimetry/methods
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Bacterial Proteins
Metal-Organic Frameworks
Endodeoxyribonucleases
Phthalic Acids
CRISPR-Associated Proteins

@article {pmid42612255,
year = {2027},
author = {Cao, Y and Ma, L and Liang, Q and Zhang, X and Ye, R and Liu, M and Xie, C and Wang, Y},
title = {Ultrasensitive and specific detection of Coxsackievirus A10 using the reverse transcription multiple cross displacement amplification combined with CRISPR/Cas12a system.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130427},
doi = {10.1016/j.talanta.2026.130427},
pmid = {42612255},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; Humans ; *Enterovirus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; *Hand, Foot and Mouth Disease/diagnosis/virology ; RNA, Viral/genetics ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {The escalating global burden of Coxsackievirus A10 (CVA10)-associated hand, foot, and mouth disease (HFMD), coupled with the lack of specific antiviral therapies, has created an urgent need for diagnostic methods that balance sensitivity, speed, and field-deployability, a balance that current molecular tools have yet to achieve. To address this gap, we developed CVA10-RT-MCDA-CRISPR analysis, an integrated platform combining reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR-Cas12a-mediated detection. Targeting the conserved VP1 of the CVA10 gene, we designed a set of MCDA primers, along with an engineered CP1 primer and a specific gRNA. The assay achieved a detection limit of 0.28 copies/μL for CVA10 RNA standards and showed no cross-reactivity with non-target pathogens. Performance was validated using 112 clinical specimens, confirming the assay's feasibility in real-world settings. Collectively, these findings establish the CVA10-RT-MCDA-CRISPR assay as a practical solution that bridges the gap between laboratory-grade sensitivity and field-ready simplicity. By integrating isothermal amplification with CRISPR-based detection in a streamlined workflow, this platform not only addresses the specific challenges of CVA10 diagnosis but also exemplifies a versatile diagnostic framework applicable to other emerging pathogens in resource-limited settings.},
}

*CRISPR-Cas Systems
Humans
*Enterovirus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Reverse Transcription
*Hand, Foot and Mouth Disease/diagnosis/virology
RNA, Viral/genetics
Sensitivity and Specificity
Limit of Detection

@article {pmid42628453,
year = {2027},
author = {Xu, L and Wang, S and Zhou, Y and Luo, J and Wang, C and Fei, Z and Wang, X},
title = {A smartphone-assisted one-tube ERA-CRISPR/Cas12a biosensor for integrated detection of hypervirulence and antimicrobial resistance determinants in Klebsiella pneumoniae.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130469},
doi = {10.1016/j.talanta.2026.130469},
pmid = {42628453},
issn = {1873-3573},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/isolation & purification/drug effects ; *Biosensing Techniques/methods ; *Smartphone ; Animals ; *Klebsiella Infections/microbiology/diagnosis ; Virulence/genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Humans ; Mice ; Rapid Diagnostic Tests ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) strains co-harboring critical antimicrobial resistance determinants pose a serious threat to public health and clinical management. Rapid identification of virulence- and resistance-associated genes is therefore essential for early diagnosis, antimicrobial stewardship, and infection control. In this study, we developed a smartphone-assisted one-tube enzymatic recombinase amplification (ERA)-CRISPR/Cas12a biosensor for simultaneous detection of four hvKP-associated virulence genes (iroB, iucA, peg-344, and rmpA) and two clinically important resistance genes (blaNDM-1 and mcr-1). To overcome the aerosol contamination risk typical of conventional CRISPR diagnostics, our platform features an innovative sealed dual-chamber reaction tube that physically isolates ERA amplification from CRISPR/Cas12a cleavage until manually punctured. The biosensor demonstrates high analytical sensitivity with a limit of detection (LOD) of 1 × 10[1] CFU/mL and can be fully completed within 45 min under isothermal conditions (42 °C). Furthermore, a custom portable fluorescence detector wirelessly transmits real-time data to a smartphone application for immediate analysis. Importantly, the biosensor exhibited 100% positive agreement with standard qPCR when evaluating blood samples from a mouse infection model and a diverse cohort of 76 clinical K. pneumoniae isolates. Overall, this portable, highly sensitive, and contamination-controlled biosensor provides a robust point-of-care testing (POCT) platform for the rapid screening of multidrug-resistant hvKP.},
}

*Klebsiella pneumoniae/genetics/pathogenicity/isolation & purification/drug effects
*Biosensing Techniques/methods
*Smartphone
Animals
*Klebsiella Infections/microbiology/diagnosis
Virulence/genetics
*CRISPR-Cas Systems
*Drug Resistance, Bacterial/genetics
Humans
Mice
Rapid Diagnostic Tests
Anti-Bacterial Agents/pharmacology

@article {pmid42659818,
year = {2027},
author = {Zhao, J and Wang, Y and Chen, B and Guo, L and Li, H and Xue, L and Xu, J and Wu, R},
title = {Synchronous dual-Cas12a activation via dimeric palindromic hairpin-programmed cascade amplification for ultrasensitive molecular diagnostics.},
journal = {Talanta},
volume = {312},
number = {Pt B},
pages = {130512},
doi = {10.1016/j.talanta.2026.130512},
pmid = {42659818},
issn = {1873-3573},
mesh = {Humans ; *Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; Inverted Repeat Sequences ; *Endodeoxyribonucleases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; *Breast Neoplasms/diagnosis/genetics ; },
abstract = {Integrating isothermal nucleic acid amplification with CRISPR/Cas12a trans-cleavage has emerged as a powerful strategy for ultrasensitive molecular diagnostics. However, most systems depend on multiple probes, separated amplification modules, or complex probe networks, increasing design complexity, optimization burden, and instability. Herein, we report a dimeric palindromic hairpin-programmed cascade amplification strategy for synchronous dual-Cas12a activation and ultrasensitive molecular diagnostics. The distinctive feature of this design lies in the construction of a single self-dimerizing palindromic hairpin (PaH) probe integrating target recognition, primer-initiated extension, nicking-site formation, cyclic trigger generation, and palindrome-directed trigger assembly. miRNA-155 was selected as a model biomarker to initiate the single-probe amplification process. Upon target recognition, the dimeric palindromic hairpin probe undergoes Phi29 polymerase-mediated extension and Nt.BbvCI-assisted cyclic nicking, continuously generating palindromic trigger strands. These triggers undergo intermolecular hybridization and polymerase-driven elongation to produce extended duplex structures containing dual crRNA-binding sites, synchronously activating two Cas12a complexes from one cascade amplification output. Owing to this architecture-embedded cascade amplification and dual-Cas12a trans-cleavage mechanism, provided a quantitative range of 1 fM to 1 nM, with a calculated detection limit of 55 aM. The assay exhibited high sequence specificity and, in a preliminary proof-of-concept evaluation using total miRNA extracts from a small cohort of healthy individuals and breast cancer patients, generated significantly different fluorescence responses between the two groups. By integrating multiple amplification and signal-transduction functions into a single probe architecture, this work provides a compact framework for constructing high-gain CRISPR/Cas12a-based biosensing systems.},
}

Humans
*Nucleic Acid Amplification Techniques/methods
*MicroRNAs/genetics
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
*Bacterial Proteins/genetics/metabolism
Inverted Repeat Sequences
*Endodeoxyribonucleases/genetics/metabolism
*Molecular Diagnostic Techniques/methods
*Breast Neoplasms/diagnosis/genetics

@article {pmid42795672,
year = {2026},
author = {Goemann, CLC and Bui, H and Rincon Miranda, S and Arnold, AD and Carlson, RP and Viamajala, S and Gerlach, R and Wiedenheft, B},
title = {Stress-Induced Metabolic Reprogramming in the Green Microalga Chlorella sp. SLA-04.},
journal = {Microorganisms},
volume = {14},
number = {9},
pages = {},
pmid = {42795672},
issn = {2076-2607},
support = {P20 GM103474/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; DE-EE0008247 and DE EE0009676//United States Department of Energy/ ; DE-AC02-05CH11231//Office of Science/ ; },
abstract = {Microalgae thrive in diverse and often challenging environments by coordinating metabolic and physiological responses to environmental stress, yet the molecular mechanisms underlying these adaptations remain incompletely understood. Here, we combined physiological, biochemical, and transcriptomic analyses to investigate the response of the green microalga Chlorella sp. SLA-04 to nitrogen depletion and high-alkalinity growth. Nitrogen depletion redirected carbon toward carbohydrate and lipid storage and was accompanied by coordinated transcriptional changes that suppressed photosynthesis while activating TAG biosynthetic and degradative pathways. The coordinated induction of lipid biosynthetic and degradative pathways is consistent with a model in which TAG synthesis and turnover help maintain redox homeostasis during nutrient stress. In contrast, high-alkalinity growth altered fatty-acid composition and was associated with transcriptional changes involving ion transport, membrane remodeling, and osmotic adaptation. Alkaline growth also altered the expression of mobile genetic elements and genes associated with RNA-mediated genome surveillance, suggesting that adaptation to high pH extends beyond central metabolism. Together, these findings provide a system-level view of how coordinated physiological and transcriptional responses enable Chlorella sp. SLA-04 to adapt to nutrient limitations and high-alkalinity growth.},
}

@article {pmid42796641,
year = {2026},
author = {Chen, Q and Xue, C and Hu, Y and Hong, Q and Liu, Z},
title = {From Molecular Recognition to Clinical Readout: Design Principles for Functional Nucleic Acid-Material Biosensors in Medical Diagnostics.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {18},
pages = {},
pmid = {42796641},
issn = {1420-3049},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nucleic Acids/chemistry ; Nucleic Acid Amplification Techniques/methods ; Nucleic Acid Hybridization ; DNA Nanostructures ; DNA, Catalytic/chemistry ; *Molecular Diagnostic Techniques/methods ; Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; },
abstract = {Functional nucleic acids can connect molecular recognition with chemical signal generation, but their diagnostic value depends on the performance of the complete sample-to-answer pathway. This critical narrative review examines representative studies published through 31 July 2026, with emphasis on recognition, amplification, material interfaces, sample preparation, readout and clinical interpretation under realistic conditions. Hybridization and ligation probes, aptamers, DNAzymes, DNA nanostructures and CRISPR-associated systems are compared by specificity, kinetics, leakage and matrix compatibility. Rolling circle amplification, hybridization chain reaction, catalytic hairpin assembly and enzymatic isothermal amplification are evaluated as reaction networks whose products must remain accessible to the selected interface. Functional materials are classified by their actual analytical role, including transduction, signal amplification, capture/enrichment, spatial organization and reagent storage. Evidence is distinguished between mechanistic studies, spiked matrices, clinical specimens, manufactured-format reproducibility and demonstrated clinical utility. We further integrate sample-to-answer workflow, assay time, complexity, regulatory considerations and clinically relevant decision thresholds. Across the literature, reliable performance depends on selective recognition before high-gain reactions, compatibility between amplification products and interfaces, explicit controls for inhibition and leakage, and validation across independent lots and representative clinical populations. These principles define a path from analytical proof of concept to reproducible and clinically interpretable diagnostic testing.},
}

*Biosensing Techniques/methods
Humans
*Nucleic Acids/chemistry
Nucleic Acid Amplification Techniques/methods
Nucleic Acid Hybridization
DNA Nanostructures
DNA, Catalytic/chemistry
*Molecular Diagnostic Techniques/methods
Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems

@article {pmid42796758,
year = {2026},
author = {Yan, S and Zhang, J and Han, Y and Jia, J and Yun, G and Liu, D and Ma, Y and Liu, Z and Wang, Y and Wang, H},
title = {A Dual-Plasmid CRISPR/Cas9-Based Genome Editing System for Efficient Gene Knockout and Large DNA Integration in Cronobacter malonaticus.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {9},
pages = {},
pmid = {42796758},
issn = {2076-0817},
support = {2025qn19//Project of the Health Commission of Xi'an City/ ; 31900121//National Natural Science Foundation of China/ ; 2024JC-YBQN-0196//Natural Science Basic Research Program of Shaanxi Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Plasmids/genetics ; *Gene Editing/methods ; *Cronobacter/genetics ; *Gene Knockout Techniques/methods ; Genome, Bacterial ; },
abstract = {Species of Cronobacter are emerging foodborne pathogens that pose a significant threat to neonates. Functional genomic studies in Cronobacter have been hindered by the lack of efficient genetic manipulation tools. Here, we established a CRISPR/Cas9-based genome editing platform for Cronobacter. We developed a dual-plasmid platform, pAmpCRISPR/pCasCm, by integrating CRISPR/Cas9 with the λ-Red recombination machinery. This platform enables scarless genome editing in Cronobacter malonaticus and proved effective for gene knockout and large-fragment integration under the tested conditions. Furthermore, we validated this platform in another clinically relevant species, Cronobacter sakazakii. The development of this genome editing toolkit provides a useful approach for fundamental research into Cronobacter pathogenesis, such as bacterial physiology studies, drug target exploration, and metabolic engineering.},
}

*CRISPR-Cas Systems
*Plasmids/genetics
*Gene Editing/methods
*Cronobacter/genetics
*Gene Knockout Techniques/methods
Genome, Bacterial

@article {pmid42796916,
year = {2026},
author = {Deligiannidou, E and Ganopoulos, I and Papasotiropoulos, V},
title = {Molecular Targets and Trait Innovation in CRISPR-Edited Vegetable Crops: An Up-to-Date Review (2020-2026).},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {18},
pages = {},
pmid = {42796916},
issn = {2223-7747},
abstract = {Vegetable crops are of fundamental nutritional and economic importance worldwide, yet their improvement through conventional breeding remains time consuming and constrained by genomic complexity. For that reason, over the period 2020-2026, CRISPR/Cas-mediated genome editing, a new genomic technique (NGT), has emerged as the leading tool for precise, rapid and targeted modification of vegetable crop genomes. In this review, we summarize selected peer-reviewed studies on CRISPR/Cas applications across a broad range of vegetable species. Tomato (Solanum lycopersicum) and potato (Solanum tuberosum) are the most edited species with the CRISPR/Cas system, with tomato being at the forefront, while other crops such as cucumber, watermelon, Brassica species, pepper, eggplant and lettuce, follow. We studied eight trait categories that have been improved, namely biotic stress resistance, abiotic stress resistance, yield and growth regulation, food/feed quality modification, color/flavor modification, storage conditions, herbicide resistance and industrial utilization. Out of these, biotic stress resistance is the most actively targeted category across both tomato and potato, with viral, fungal, bacterial and oomycete pathogens being addressed through the editing of host susceptibility genes. Some advances include editing SlDMR6-1 and DMR6 orthologs to confer broad-spectrum disease resistance, multiplex editing of the MLO gene family to achieve powdery mildew resistance, the utilization of the CRISPR/Cas13 system to confer resistance in RNA viruses in potato, and the engineering of drought-tolerant and quality-improved varieties across multiple species. Finally, the regulatory landscape for genome-edited products in the European Union is currently evolving with efforts to distinguish them from genome-modified products and drive further growth for the field.},
}

@article {pmid42797803,
year = {2026},
author = {Iqbal, U and Khalid, K and Shaltout, M and Yukselten, Y and Sutton, RE},
title = {CCR5 as a Therapeutic Target in HIV Disease: From CRISPR/Cas9 Gene Editing to Maraviroc-Mediated Inhibition.},
journal = {Viruses},
volume = {18},
number = {9},
pages = {},
pmid = {42797803},
issn = {1999-4915},
mesh = {Humans ; *Maraviroc/therapeutic use/pharmacology ; *HIV Infections/drug therapy/therapy/virology/genetics ; *Receptors, CCR5/genetics/metabolism ; *Gene Editing ; *CCR5 Receptor Antagonists/therapeutic use/pharmacology ; HIV-1/drug effects/physiology ; *CRISPR-Cas Systems ; Animals ; Host-Directed Therapy ; },
abstract = {The C-C chemokine receptor type 5 (CCR5) is the principal co-receptor for R5-tropic HIV-1 and remains one of the most promising therapeutic targets in the pursuit of an HIV cure. The discovery that individuals carrying the naturally occurring CCR5Δ32 mutation exhibit marked resistance to HIV infection established the foundation for both genetic and pharmacological approaches to CCR5 inhibition. This review summarizes recent advances in CCR5-targeted therapies with a focus on CRISPR/Cas9-mediated gene editing and maraviroc-mediated receptor blockade. We discuss the molecular mechanisms, preclinical evidence and emerging clinical data supporting CRISPR-based CCR5 disruption, including multiplex editing strategies designed to overcome viral tropism switching. We also examine the evolving role of maraviroc beyond viral entry inhibition, highlighting its immunomodulatory effects, potential latency-reversing activity and applications in graft-versus-host disease and cancer. Together, these complementary strategies underscore the potential of CCR5-targeted interventions as integral components of future combination therapies aimed at achieving durable HIV remission or functional cure.},
}

Humans
*Maraviroc/therapeutic use/pharmacology
*HIV Infections/drug therapy/therapy/virology/genetics
*Receptors, CCR5/genetics/metabolism
*Gene Editing
*CCR5 Receptor Antagonists/therapeutic use/pharmacology
HIV-1/drug effects/physiology
*CRISPR-Cas Systems
Animals
Host-Directed Therapy

@article {pmid42798769,
year = {2026},
author = {Yi, W and Luo, H and Luo, F and Li, Q and Huang, X and Yu, Q and Deng, G and Liu, S},
title = {CRISPR/Cas diagnostics for Klebsiella pneumoniae: from resistance and hypervirulence markers to clinically interpretable risk reporting.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1949504},
pmid = {42798769},
issn = {2296-4185},
abstract = {Klebsiella pneumoniae (Kp) is a major cause of healthcare-associated and community-onset invasive infections. The emergence of carbapenem-resistant Kp (CRKP), hypervirulent Kp (hvKp), and resistance-hypervirulence convergence has increased the need for rapid tests that provide organism context and clinically interpretable risk information. CRISPR/Cas diagnostics combine programmable sequence recognition with flexible signal generation and can be integrated with isothermal amplification, portable readouts, and multiplex workflows. Available studies support the analytical feasibility of detecting Kp- or Klebsiella pneumoniae species complex (KpSC)-associated organism-context targets, major resistance genes, and hypervirulence-associated markers, but clinical and workflow validation remains limited because many studies use small cohorts, cultured isolates, or spiked matrices. Here, clinically interpretable risk reporting means translating validated molecular findings into bounded categories-organism context, gene-associated resistance risk, hypervirulence-associated risk, and suspected resistance-hypervirulence convergence risk-while stating interpretive limits and appropriate confirmatory actions. We critically evaluate CRISPR/Cas engineering, target selection, multiplexing, specimen-specific workflows, and validation requirements, and propose a conceptual four-layer target-to-report framework for prospective evaluation. CRISPR/Cas-based Kp testing should complement, rather than replace, culture, phenotypic antimicrobial susceptibility testing, virulence assessment, and genomic confirmation.},
}

@article {pmid42635115,
year = {2026},
author = {Yang, G and Chen, Y and Zhao, W and Zhong, H},
title = {One-pot SP-EXPAR-integrated CRISPR/Cas14a assay for highly specific and versatile detection of ctDNA mutations in lung cancer patients.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {37},
pages = {8273-8280},
doi = {10.1039/d6ay01540a},
pmid = {42635115},
issn = {1759-9679},
mesh = {Humans ; *Lung Neoplasms/genetics/blood/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Circulating Tumor DNA/genetics/blood ; *CRISPR-Cas Systems/genetics ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/blood ; },
abstract = {The highly specific and versatile detection of KRAS mutations in circulating tumor DNA (ctDNA) from plasma has critical clinical implications for non-small-cell-lung cancer (NSCLC). However, conventional isothermal amplification methods suffer from poor single-base discrimination, while CRISPR-12a-based detection is highly protospacer adjacent motif (PAM)-dependent. To address these challenges, a one-pot self-primer isothermal exponential amplification reaction (SP-EXPAR) combined with a CRISPR/Cas14a assay was developed for detecting KRAS G12C and G12D. Two synergistic strategies were devised to ensure high specificity: first, a carefully designed hairpin probe that permits selective amplification of mutant over wild-type sequences through differential binding affinity; second, optimization of the Cas14a sgRNA seed region, with the mutation positioned at the 11th nucleotide for stringent target recognition. The assay is further distinguished by a physical separation design, in which the Cas14a reagents are pre-loaded into the tube cap and mixed with the amplification products only after SP-EXPAR completion. This assay enables KRAS G12C detection within 1 h, with a limit of detection of 81.9 aM (0.1% mutation percentage) and a dynamic range from 100 aM to 1 nM. Furthermore, this assay further demonstrates its programmability and was successfully applied to detect KRAS G12D with comparable performance. In detecting 42 clinical samples, this assay demonstrated 100% sensitivity and 100% specificity compared with DNA sequencing. This approach holds great potential in disease diagnosis.},
}

Humans
*Lung Neoplasms/genetics/blood/diagnosis
*Nucleic Acid Amplification Techniques/methods
*Circulating Tumor DNA/genetics/blood
*CRISPR-Cas Systems/genetics
Mutation
Proto-Oncogene Proteins p21(ras)/genetics
*Carcinoma, Non-Small-Cell Lung/genetics/blood

@article {pmid42720610,
year = {2026},
author = {Mukherjee, S and Karati, D and Sarkar, S and Ash, D and Ganguly, SC and Dutta, A and Kesarla, R},
title = {CRISPR-Cas9‑based lipid nanocarriers for advanced therapy of urinary bladder cancer.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {18},
pages = {2695-2705},
doi = {10.1080/17435889.2026.2732014},
pmid = {42720610},
issn = {1748-6963},
mesh = {Humans ; *Urinary Bladder Neoplasms/therapy/genetics/drug therapy ; *CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; *Nanoparticles/chemistry ; Gene Editing/methods ; Animals ; Genetic Therapy/methods ; Drug Carriers/chemistry ; },
abstract = {Bladder cancer (BCa) exhibits significant genetic and phenotypic variability. This variability suggests that various tumor subtypes could be influenced by several biomarkers and signaling pathways, which presents a problem for monotherapy strategies. Despite the initial effectiveness of traditional therapies, BCa's high rates of progression and recurrence, and the eventual development of drug resistance in many patients, continue to be major concerns. Because of the potential to transform the genetic modifications linked to the disease, genome editing using CRISPR/Cas9 has become a transformative tool in medicine with noteworthy potential for BCa therapy. Although the CRISPR/Cas9 technology is incredibly effective at transforming the field of genome editing, its instability and cellular impermeability pose significant challenges to its delivery. To increase efficient delivery of CRISPR/Cas9, nanovectors may be investigated. Significant promise exists for improving the therapeutic potential of CRISPR-Cas9 technology and addressing complex cancer therapy difficulties because of the rapid development of nanotechnology-based delivery systems. Relevant articles were searched in Google Scholar, Scopus, and Web of Science covering studies published between 2007 and 2026. Along with the impact of lipid-based nanoparticles on their safe transport to cancer cells, this review emphasizes the significance of the CRISPR/Cas9 genome editing system in the treatment of BCa.},
}

Humans
*Urinary Bladder Neoplasms/therapy/genetics/drug therapy
*CRISPR-Cas Systems/genetics
*Lipids/chemistry
*Nanoparticles/chemistry
Gene Editing/methods
Animals
Genetic Therapy/methods
Drug Carriers/chemistry

@article {pmid42788817,
year = {2026},
author = {Ganesan, BK and Mishra, A and Hota, D and Maiti, R},
title = {Non-antibiotic therapeutic approaches for antimicrobial resistance: current evidence and future directions.},
journal = {Expert review of clinical pharmacology},
volume = {},
number = {},
pages = {1-17},
doi = {10.1080/17512433.2026.2739560},
pmid = {42788817},
issn = {1751-2441},
abstract = {INTRODUCTION: Antimicrobial resistance (AMR) is a global health challenge that reduces the effectiveness of existing antibiotics, leading to increased morbidity, mortality and healthcare costs worldwide. The lack of development of novel classes of antibiotics has led to an increased interest in non-antibiotic therapeutic approaches that focus on bacterial virulence, host-pathogen interactions, microbial ecology, and resistance mechanisms.

AREAS COVERED: This review summarizes the current evidence on non-antibiotic therapeutics including bacteriophages, antimicrobial peptides, anti-virulence agents, monoclonal antibodies, microbiome-based therapies, CRISPR-Cas systems, nanoparticles, photodynamic therapy, repurposed non-antibiotic drugs and combination strategies. We performed a literature search on PubMed, Embase, Scopus, Web of Science, and Google Scholar until May 2026. Though several approaches have demonstrated promising biological activity and favourable safety profiles, clinical evidence remains limited and heterogeneous.

EXPERT OPINION: Non-antibiotic therapeutics are vital adjunctive, salvage and precision approaches to the challenge of AMR. However, most strategies are still at an early translational stage with a paucity of high-quality randomized clinical evidence. Barriers include delivery, manufacturing complexity, regulatory uncertainty, cost, and lack of long-term safety data. Further progress will depend on standardization of production, better delivery platforms, well-designed multicentric clinical trials, and incorporation into antimicrobial stewardship and precision medicine frameworks.},
}

@article {pmid42790056,
year = {2026},
author = {Yang, H and Yi, X and Wang, X and Wen, H and Zhang, L and Ding, M and Wu, S and Wang, Y and Wang, M and Dong, L and Wang, X and Peng, D},
title = {Programmable nuclease-based biosensors for food safety: Recent advances and perspectives.},
journal = {Talanta},
volume = {313},
number = {Pt A},
pages = {130662},
doi = {10.1016/j.talanta.2026.130662},
pmid = {42790056},
issn = {1873-3573},
abstract = {Programmable nucleases are emerging as powerful tools for rapid and sensitive food contaminant detection. This review systematically examines five representative nucleases-Cas9, Cas12, Cas13, Cas14, and CbAgo-with emphasis on their target-recognition mechanisms, signal-transduction pathways, amplification strategies, and readout formats. The distinct analytical strengths of Cas9, Cas12, Cas13, and Cas14 are compared in terms of specificity, collateral cleavage, multiplexing capability, and suitability for nucleic acid and non-nucleic acid targets. Particular attention is given to Cas14 and CbAgo, two less extensively reviewed nucleases with PAM-independent recognition and promising potential for flexible target selection and multiplex sensing. In addition, recent advances in artificial intelligence and machine vision for fluorescence image processing, digital counting, and multiplex quantification are critically discussed. Finally, current challenges and future priorities are outlined, including one-pot integration, signal amplification, assay standardization, miniaturization, and field deployment. This review provides a comparative framework for developing practical programmable nuclease-based food-safety platforms.},
}

@article {pmid42790585,
year = {2026},
author = {Chen, R and Yan, Y and Yan, S and Chen, L and Yu, Y and Wang, Y},
title = {Mechanisms underlying host-specific horizontal transfer of shrimp AHPND virulence plasmid: Vibrio immunity and colonization environment.},
journal = {Journal of invertebrate pathology},
volume = {220},
number = {},
pages = {108755},
doi = {10.1016/j.jip.2026.108755},
pmid = {42790585},
issn = {1096-0805},
abstract = {Acute hepatopancreatic necrosis disease (AHPND) is a highly contagious and lethal shrimp disease caused by Vibrio species harboring the virulence plasmid pAHPND, which encodes pirAB toxin genes. Here, we employed comprehensive comparative genomic and bioinformatic analyses to elucidate the mechanisms that govern the host-specific horizontal transfer of pAHPND. Our analyses revealed that although the majority of Vibrio host strains harboring pAHPND or pAHPND-related plasmids (71%, 27/38) possessed restriction-modification (R-M) systems, these plasmids encode anti-restriction proteins (ArdC, KlcA), suggesting R-M systems alone may be insufficient to block plasmid dissemination. Of the 92 non-AHPND Vibrio strains with CRISPR-Cas systems targeting pAHPND or its related plasmids, 64 specifically targeted the pAHPND plasmid itself. The primary targets within this plasmid were genes involved in fimbriae biosynthesis. Analysis of antimicrobial resistance genes (ARGs) in these CRISPR-positive strains showed that half (46/92) carried more than five ARGs, indicating robust environmental adaptability. In contrast, Vibrio strains that stably maintained pAHPND lacked CRISPR spacers targeting this plasmid and carried fewer ARGs (with 76% of strains harboring no more than seven ARGs). Collectively, these results suggest that the dissemination of pAHPND is shaped by Vibrio immune defenses - particularly CRISPR-Cas systems - and by selective pressures in the colonization environment, including antibiotic use. These findings highlight the evolutionary interplay between bacterial immunity, environmental stress, and virulence plasmid persistence.},
}

@article {pmid42793032,
year = {2026},
author = {Ruden, DM},
title = {Prime Editing for Precision Genetic Medicine: A Systematic Review of Technologies, Delivery, and Therapeutic Applications.},
journal = {Genes},
volume = {17},
number = {9},
pages = {},
pmid = {42793032},
issn = {2073-4425},
support = {P42 ES030991/ES/NIEHS NIH HHS/United States ; 5P42ES030991-05P42/NH/NIH HHS/United States ; UG3 OD023285/OD/NIH HHS/United States ; P30 ES036084/ES/NIEHS NIH HHS/United States ; 1P30ES036084-03P30/NH/NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Precision Medicine/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Transfer Techniques ; },
abstract = {BACKGROUND: Prime editing has rapidly evolved from a CRISPR-based "search-and-replace" approach for precise sequence modification into a diverse family of genome editing technologies. This systematic review maps the technological evolution of prime editing, with emphasis on editor architecture, guide RNA engineering, delivery, therapeutic applications, computational approaches, and emerging capabilities.

METHODS: PubMed and Web of Science were systematically searched for studies in which prime editing constituted a substantive experimental, technological, computational, delivery, or therapeutic component. After deduplication and screening, candidate studies underwent manual re-screening against prespecified eligibility criteria. Reviews, corrections, plant and bacterial studies, conventional CRISPR or base editing studies without a substantive prime editing component, and other non-relevant records were excluded. A total of 294 studies were included in the final systematic evidence synthesis. Because of substantial heterogeneity in editor architectures, targets, experimental models, outcomes, and reporting, the literature was synthesized using systematic mapping and qualitative thematic analysis rather than meta-analysis.

RESULTS: The evidence demonstrates rapid diversification from the original Cas9 nickase-reverse transcriptase-prime editing guide RNA architecture through improvements in pegRNA design, Cas and reverse transcriptase engineering, DNA repair modulation, delivery, computational design, and increasingly complex sequence modification. Therapeutic studies span disease modeling, correction of pathogenic variants, ex vivo applications, and direct in vivo editing; however, high editing efficiency does not necessarily translate into functional or therapeutic rescue. Large-sequence insertion and replacement strategies further extend the capabilities of prime editing, although these approaches remain less mature than small-sequence correction and face substantial challenges in efficiency, fidelity, cargo delivery, and genomic safety.

CONCLUSIONS: Prime editing has developed into a versatile precision genome editing platform, but the evidence base remains heterogeneous and predominantly preclinical. Translation to genetic medicine will require improvements in reproducibility across targets and cell types, delivery to clinically relevant tissues, product purity, genomic safety, and demonstration of meaningful functional benefit. Emerging large-sequence editing approaches broaden the potential scope of prime editing but should be distinguished from technologies with established experimental and therapeutic evidence.},
}

Humans
*Gene Editing/methods
*Precision Medicine/methods
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Animals
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Transfer Techniques

@article {pmid42793101,
year = {2026},
author = {Kim, MS and Do, HS and Jang, HY and Lee, KE and Lee, SJ},
title = {Recent Advances in CRISPR/Cas Technologies for Biological Discovery, Therapeutics, and Diagnostics.},
journal = {Biomolecules},
volume = {16},
number = {9},
pages = {},
pmid = {42793101},
issn = {2218-273X},
support = {RS-2026-25481309//National Research Foundation of Korea/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Neoplasms/therapy/genetics/diagnosis ; Animals ; Genetic Therapy/methods ; Precision Medicine ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system began as a tool for programmable genome editing. CRISPR/Cas technologies have evolved into a versatile platform for functional genomic screening, epigenome editing, therapeutic target discovery, and highly sensitive molecular diagnostics. New effectors and engineered variants continue to push these applications into personalized medicine and point-of-care testing. Here, this review highlights recent advances in therapeutic target discovery and therapeutic and molecular diagnostic development utilizing CRISPR/Cas technologies. We discuss how CRISPR interference (CRISPRi), CRISPR activation (CRISPRa), base editing, and prime editing have improved our understanding of disease mechanisms, while creating new opportunities for therapeutic intervention. Current applications in cancer immunotherapy, infectious disease, and neurological disorders are also discussed. In diagnostics, CRISPR-based platforms enable sensitive detection of infectious pathogens, cancer biomarkers, and genetic disorders using programmable nuclease activity in both laboratory and point-of-care settings. Collectively, these advances continue to expand the role of CRISPR/Cas technology across biological discovery, disease diagnostics, and therapeutic development, driving progress in precision medicine.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Neoplasms/therapy/genetics/diagnosis
Animals
Genetic Therapy/methods
Precision Medicine

@article {pmid42793135,
year = {2026},
author = {Hale, A and Nusawardhana, A and Nicolae, CM and Moldovan, GL},
title = {Genome-Wide CRISPR Screens Identify Genome Stability as Regulator of the Cellular Sensitivity to Environmentally Relevant Bisphenol A Exposure.},
journal = {Biomolecules},
volume = {16},
number = {9},
pages = {},
pmid = {42793135},
issn = {2218-273X},
support = {R01 ES026184/ES/NIEHS NIH HHS/United States ; R01 GM134681/GM/NIGMS NIH HHS/United States ; },
mesh = {*Benzhydryl Compounds/toxicity ; Bisphenol A Compounds ; *Phenols/toxicity ; Humans ; *Genomic Instability/drug effects/genetics ; HeLa Cells ; DNA Damage/drug effects ; *CRISPR-Cas Systems ; DEAD-box RNA Helicases/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Bisphenol A (BPA) is a prevalent chemical used in the production of plastics. While adverse effects on the reproductive system have been documented, more recent studies also associated BPA exposure with carcinogenesis as well as genomic instability. However, these studies were generally performed using BPA concentrations much higher than those observed in the serum or urine of the general population, making their relevance unclear. To address this, we report here an unbiased genetic study to identify mechanisms responding to levels of BPA exposure relevant to plastic manufacturers. We performed genome-wide CRISPR knockout screens in HeLa and RPE1 cells upon continuous exposure to 0.5 μM BPA, a concentration similar to the mean BPA concentration found in the urine of plastics manufacturing workers, for 19 days. We found genome stability genes among the top common hits between the two cell lines, suggesting that BPA causes DNA damage at this environmentally relevant exposure dose. We validated the DNA repair gene RAD51C and the RNA helicase DDX21 as genes required for BPA resistance. Our study suggests that BPA exposure at environmentally relevant doses can cause DNA damage, highlighting the relevance of BPA for carcinogenesis.},
}

*Benzhydryl Compounds/toxicity
Bisphenol A Compounds
*Phenols/toxicity
Humans
*Genomic Instability/drug effects/genetics
HeLa Cells
DNA Damage/drug effects
*CRISPR-Cas Systems
DEAD-box RNA Helicases/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42793231,
year = {2026},
author = {Li, J and Zhang, X},
title = {Research Progress in Applications of the CRISPR-Cas12b System in Pathogen Nucleic Acid Detection.},
journal = {Current issues in molecular biology},
volume = {48},
number = {9},
pages = {},
pmid = {42793231},
issn = {1467-3045},
support = {32360889//National Natural Science Foundation of China/ ; 2026-NK-Z47//he Special Project for Scientific and Technological Achievement Transformation of the Central Government Guided Local Science and Technology Development Fund of Qinghai Provincial Department of Science and Technology/ ; },
abstract = {This review systematically summarizes recent advances in CRISPR-Cas12b-based pathogen nucleic acid detection. Starting with an overview of conventional nucleic acid detection methods and core CRISPR-Cas mechanisms, we highlight the unique properties that distinguish Cas12b from other Cas effectors. We elaborate on the working principles of the CRISPR-Cas12b system and present comparative structural and functional analyses with other Cas variants to underscore its distinctive molecular architecture and enzymatic properties. A comprehensive evaluation of current applications demonstrates the efficacy of CRISPR-Cas12b-based diagnostics across diverse pathogens, including viruses, bacteria, and parasites, with a specific focus on its integration with isothermal amplification techniques. We further examine the translational potential of CRISPR-based diagnostics in clinical settings, while critically analyzing persistent technical challenges including off-target effects, signal amplification limitations, and sample preparation requirements. Targeted strategic recommendations are proposed to optimize detection sensitivity, develop multiplexed detection platforms, and implement point-of-care testing configurations. This review aims to systematically correlate the unique characteristics of Cas12b with its broad diagnostic applications, thereby addressing key research gaps in its progression toward clinical validation and field deployment. It also provides a targeted framework to accelerate the translation of this technology from laboratory platforms to practical diagnostic solutions.},
}

@article {pmid42793835,
year = {2026},
author = {Mukhametova, LI and Eremin, SA and Tsakalof, AK},
title = {Disease Biomarker Detection Using Fluorescence Polarization Assays: Principles and Clinical Applications.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {16},
number = {18},
pages = {},
pmid = {42793835},
issn = {2075-4418},
support = {1210415000398//Lomonosov Moscow State University/ ; },
abstract = {Modern clinical diagnostics require highly sensitive, rapid, and technologically simple methods for quantitatively determining biomarkers directly at the patient's bedside. Fluorescence polarization (FP) meets these criteria, as it enables the homogeneous recording of an analytical signal based on changes in the rotational diffusion of a fluorescently labeled ligand upon binding to a target, without separation or washing steps. This comprehensive narrative review analyzes the literature over the past 10-15 years devoted to the use of FP analysis for detecting biomarkers of socially significant diseases. The fundamental principles of the method and modern analysis formats are discussed-from classical fluorescence polarization immunoassay (FPIA) with antibodies to aptamer sensors, DNAzyme-based systems, and reagent-free biosensors such as Quenchbody. Particular attention is paid to new approaches to signal amplification, including those using nanoparticles, protein aggregation, and isothermal amplification of nucleic acids. The main part of the review is devoted to practical applications of FP: diagnostics of infectious diseases (brucellosis, tuberculosis, and viral infections) demonstrating sensitivity at the level of traditional ELISA; liquid biopsy in oncology, including the detection of extracellular vesicles using aptamer FP platforms; therapeutic drug monitoring of antibiotics in whole blood on portable paper media; and the identification of biomarkers for metabolic and ophthalmological disorders. Prospects for integrating polarization detection with CRISPR/Cas systems, microfluidics, and smartphone-compatible readers are discussed, paving the way to the creation of a new generation of inexpensive devices for personalized medicine.},
}

@article {pmid42794534,
year = {2026},
author = {Mamaeva, NY and Yakovlev, VA and Kristovskiy, NV and Feskin, PG and Vinnikov, RS and Oleinikov, PD and Shmakov, SA and Shaitan, KV and Kirpichnikov, MP and Shaytan, AK},
title = {3D Genome Engineering Using CRISPR/dCas Systems.},
journal = {International journal of molecular sciences},
volume = {27},
number = {18},
pages = {},
pmid = {42794534},
issn = {1422-0067},
support = {075-15-2025-475//The Ministry of Education and Science of the Russian Federation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; Chromatin/genetics ; Epigenome Editing ; *Gene Editing/methods ; *Genome ; *Genetic Engineering/methods ; Epigenesis, Genetic ; },
abstract = {The spatial organization of the genome has emerged as a central regulator of gene expression and cellular function. Chromatin architecture is organized hierarchically across multiple spatial scales and involves chromatin loops, topologically associating domains (TADs), chromatin compartments, and specialized nuclear environments that collectively shape regulatory interactions within the nucleus. Disruption of these structures contributes to a wide range of diseases, including developmental disorders, cancer, and laminopathies, stimulating growing interest in technologies capable of programmable manipulation of genome topology. The emergence of CRISPR/dCas-based technologies has transformed the field from descriptive 3D genomics to programmable genome engineering. Catalytically inactive Cas proteins fused to architectural or epigenetic effectors enable targeted manipulation of chromatin loops, loop extrusion, subnuclear positioning, and local chromatin states without altering the underlying DNA sequence. In this review, we summarize current CRISPR/dCas-based approaches for engineering three-dimensional genome architecture, discuss their mechanistic basis and applications, and highlight emerging therapeutic opportunities and major technical challenges in the field.},
}

Humans
*CRISPR-Cas Systems
Animals
Chromatin/genetics
Epigenome Editing
*Gene Editing/methods
*Genome
*Genetic Engineering/methods
Epigenesis, Genetic

@article {pmid42794786,
year = {2026},
author = {Fan, S and Liang, R and Fei, T},
title = {CRISPR Technologies in Type 2 Diabetes: From Mechanistic Insights to Therapeutic Discovery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {18},
pages = {},
pmid = {42794786},
issn = {1422-0067},
support = {2025YFA0921900//National Key Research and Development Program of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; *Diabetes Mellitus, Type 2/genetics/therapy/metabolism ; Animals ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Insulin-Secreting Cells/metabolism ; *Gene Editing/methods ; Lipid Metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; Glucose/metabolism ; Genetic Predisposition to Disease ; },
abstract = {Type 2 diabetes (T2D) is a complex metabolic disorder driven by the interplay of genetic susceptibility, β-cell dysfunction, and insulin resistance. Although genome-wide association studies (GWAS) have identified numerous T2D risk loci, translating these genetic findings into biological mechanisms and therapeutic strategies remains challenging. The emergence of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based technologies has provided powerful tools for precise genome manipulation and functional interrogation of disease-associated genes and genetic variations. This review summarizes the development of CRISPR technologies and highlights their applications in T2D research. We review the application of CRISPR technologies in T2D genetic factor analysis and high-throughput phenotypic screening, with a focus on three key areas: glucose metabolism, lipid metabolism, and pancreatic functionality. We further examine the role of CRISPR in therapeutics that target pancreatic β-cells via gene correction, stress protection, and epigenetic reprogramming. Finally, we briefly discuss the major challenges encountered in the applications of CRISPR technology, including delivery efficiency, off-target effects, safety concerns and ethical considerations.},
}

Humans
*Diabetes Mellitus, Type 2/genetics/therapy/metabolism
Animals
*CRISPR-Cas Systems
*Genetic Therapy/methods
Insulin-Secreting Cells/metabolism
*Gene Editing/methods
Lipid Metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome-Wide Association Study
Glucose/metabolism
Genetic Predisposition to Disease

@article {pmid41718579,
year = {2026},
author = {Shih, J and Yokomi, RK and Hajeri, S and Roy, A and Wei, W},
title = {Field-Compatible Detection of Spiroplasma citri Associated with Citrus Stubborn Disease Using CRISPR-Cas12a and Crude Sample Preparation.},
journal = {Plant disease},
volume = {110},
number = {9},
pages = {3694-3702},
doi = {10.1094/PDIS-08-25-1699-RE},
pmid = {41718579},
issn = {0191-2917},
mesh = {*Citrus/microbiology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Spiroplasma citri/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; },
abstract = {Citrus stubborn disease (CSD), caused by Spiroplasma citri, presents a significant risk to citrus production, resulting in considerable yield losses when infections remain undetected. Accurate and timely diagnosis is crucial for effective disease management. However, existing nucleic acid-based methods, such as PCR and quantitative PCR (qPCR), require laboratory equipment and are not easily applicable in the field. This study developed a CRISPR-Cas12a-based DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) assay for the rapid, sensitive, and specific detection of S. citri, targeting the spiralin gene. An optimized recombinase polymerase amplification (RPA) primer pair and CRISPR-RNA (crRNA) were utilized for sequence-specific activation of Cas12a, enabling cleavage of fluorescent and lateral flow-compatible reporters. The assay demonstrated a detection limit of 1 attomolar (aM) (around 1.8 genome copies) using a fluorescence plate reader and 10 aM using blue-light visualization and a lateral flow assay (LFA). Specificity testing revealed discrimination against other phytopathogenic spiroplasmas, including S. kunkelii and S. melliferum. Validation in the plate-reader format with DNA extracted from symptomatic citrus samples showed 100% consistency with qPCR results. A 10-min NaOH-Tris crude extraction protocol was also assessed, facilitating straightforward and equipment-free sample preparation. Relative to DETECTR assays with kit-extracted samples, crude extracts preserved full diagnostic sensitivity in fluorescence assays and achieved 70% accuracy in LFA and visual formats in a subset of the same samples. These findings establish a dependable, portable, and highly sensitive diagnostic approach for S. citri, providing a practical tool for on-site detection and enhanced management of citrus stubborn disease.},
}

*Citrus/microbiology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology
*Spiroplasma citri/genetics/isolation & purification
CRISPR-Associated Proteins/genetics
Bacterial Proteins/genetics
Rapid Diagnostic Tests
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases

@article {pmid41933454,
year = {2026},
author = {Ying, XY and Tang, DL and Sun, YM and Liu, MQ and Feng, XY and Wang, FY and Lu, C and Luo, ZD},
title = {Soil In Situ Enrichment Coupled with RPA-CRISPR/Cas12b for Rapid and Visual On-Site Detection of Fusarium oxysporum in Strawberry.},
journal = {Plant disease},
volume = {110},
number = {9},
pages = {4013-4021},
doi = {10.1094/PDIS-01-26-0110-RE},
pmid = {41933454},
issn = {0191-2917},
mesh = {*Fragaria/microbiology ; *Fusarium/isolation & purification/genetics ; *Soil Microbiology ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Soil/chemistry ; },
abstract = {Fusarium oxysporum is a representative soilborne fungal pathogen that causes strawberry wilt, a disease characterized by an extremely high mortality rate that poses a severe threat to the sustainable development of the global strawberry industry. However, traditional detection methods are often time-consuming and dependent on specialized laboratory equipment, while existing soil nucleic acid extraction protocols are highly susceptible to interference from inhibitors in complex matrices, leading to low detection efficiency or high false-negative rates. To address these limitations, this study developed a novel on-site detection platform based on in situ biological enrichment and purification-free nucleic acid release. A specialized enrichment rod targeting F. oxysporum was developed to leverage the tropic growth characteristics of the pathogen, achieving physical separation from the soil matrix and effectively eliminating interference from complex soil inhibitors such as humic acids. The enriched pathogens release nucleic acids via a rapid lysis buffer, which are then neutralized and used directly as templates for recombinase polymerase amplification (RPA)-CRISPR/Cas12b isothermal detection, enabling visual field identification through lateral flow strips. This method requires no specialized instrumentation, achieving a detection limit of 8.5 CFU/g and a sensitivity of 70 copies. After completing 48 h of in situ enrichment, the entire process from rod retrieval to detection completion requires only 40 min (with hands-on operation time <10 min). By effectively circumventing soil inhibitor interference and simplifying complex nucleic acid extraction into a rapid, integrated protocol, this platform provides a critical technical solution for the on-site monitoring and precise control of soilborne pathogens.},
}

*Fragaria/microbiology
*Fusarium/isolation & purification/genetics
*Soil Microbiology
*Plant Diseases/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Soil/chemistry

@article {pmid42628204,
year = {2026},
author = {Tanveer, A and Khan, SH and Atif, RM and Azhar, MT and Rehman, HM and Rana, IA and Yang, SH},
title = {Blocking the conversion of β-carotenoids into xanthophylls through HYb gene editing via CRISPR/Cas9 confers enhanced heat stress tolerance in Nicotiana tabacum.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111638},
doi = {10.1016/j.plaphy.2026.111638},
pmid = {42628204},
issn = {1873-2690},
mesh = {*Nicotiana/genetics/metabolism/physiology ; *Xanthophylls/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *beta Carotene/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Heat-Shock Response/genetics ; Chlorophyll/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Plants produce protective metabolites to withstand stress, and β-carotenoids act as crucial antioxidants. β-Carotene is converted into xanthophylls by β-carotene hydroxylase (BCH). In this study, the BCH gene in Nicotiana tabacum was targeted using CRISPR/Cas9 genome editing to redirect metabolic flux toward β-carotene accumulation and evaluate its effects on heat tolerance. Following Agrobacterium-mediated transformation with a BCH-specific gRNA, three mutant plants were obtained; two carrying frameshift mutations (P1 and P3) and one with mis-sense mutations (P2). A significant increase in chlorophyll contents was observed in the edited lines compared with wild-type plants. Interestingly, the frameshift mutants exhibited substantial increase in total chlorophyll (71% and 31% in P3 and P1 respectively) as compared to mis-sense mutant P2 (18%). Further, biochemical profiling using HPLC showed that the edited plants accumulated 3.74-fold increase in β-carotene than wild-type plants. Under heat stress (42 °C for 96 h), the edited plants exhibited enhanced thermotolerance by remaining greener and delayed wilting, whereas wild-type plants developed necrosis rapidly under heat stress. This enhanced thermotolerance might has resulted from the increased expression of ε-cyclase (an upstream key enzyme in β-carotene biosynthesis) as revealed by expression profiling of edited plants. In contrast, the expression of downstream β-cryptoxanthin and zeaxanthin biosynthetic genes was reduced, which seems consistent with mutation of BCH. DAB staining further confirmed reduced ROS accumulation in edited plants as compare to wild type supporting the protective role of β-carotene. In summary, the downregulation of BCH in N. tabacum boosted β-carotene levels and chlorophyll retention, enhancing heat stress tolerance. These findings demonstrate the potential of targeting carotenoid biosynthesis to improve crop resilience.},
}

*Nicotiana/genetics/metabolism/physiology
*Xanthophylls/metabolism
*Mixed Function Oxygenases/genetics/metabolism
*beta Carotene/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
*Heat-Shock Response/genetics
Chlorophyll/metabolism
Gene Expression Regulation, Plant

@article {pmid42679775,
year = {2026},
author = {Li, S and Liao, Y and Wang, X and Chen, G and Liu, Y and Pan, S and Zhen, T and Li, X and Zhu, F and Qin, R},
title = {Double-end blocker and split input mediated CRISPR-Cas12a system for modular single-base mutation detection with low abundance.},
journal = {Biosensors & bioelectronics},
volume = {314},
number = {},
pages = {119176},
doi = {10.1016/j.bios.2026.119176},
pmid = {42679775},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Pancreatic Neoplasms/genetics/diagnosis ; *Proto-Oncogene Proteins p21(ras)/genetics ; DNA Mutational Analysis/methods ; Mutation ; Limit of Detection ; },
abstract = {Traditional CRISPR-Cas12a mutation detection systems are limited by poor single-base specificity, target-specific crRNA redesign, and insufficient sensitivity for low-abundance mutations, restricting their clinical liquid biopsy applications. Herein, we developed a crRNA-universal, sensitive and specific CRISPR-Cas12a detection platform, termed DESIC (double-end blocker and split-input mediated CRISPR-Cas12a system), for single-base mutation detection. The DESIC system adopts two key structural designs: double-end blocker (DEB) and duplicated split-input (SIN). The DEB spatially isolates crRNA recognition and target-binding regions, enabling universal detection of various mutation sites without crRNA redesign. The SIN strategy amplifies thermodynamic differences from single-base mismatches, greatly improving single-nucleotide discrimination. We targeted four prevalent pancreatic cancer KRAS mutations (G12D, G12R, G12V, Q61H) and optimized the system to achieve optimal discrimination. The optimized DESIC system exhibited ultra-low limits of detection down to 0.01% mutant allele fraction with reliable linear quantitative performance. Clinical validation using 15 pairs of pancreatic cancer tissue and peripheral blood samples confirmed that DESIC results were highly consistent with gold-standard NGS data. With a flexible modular design, this low-cost, easy-operated platform can be readily extended to multiple tumor mutations, holding great potential for tumor liquid biopsy and early molecular diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Pancreatic Neoplasms/genetics/diagnosis
*Proto-Oncogene Proteins p21(ras)/genetics
DNA Mutational Analysis/methods
Mutation
Limit of Detection

@article {pmid42696818,
year = {2026},
author = {Zhang, S and Xie, S and Wang, J and Zhang, X and Cai, Y and Lai, X and Min, N and Qu, J and Peng, B and Li, T and Zhang, J and Yang, D and Shi, X and Xia, N and Ge, S},
title = {Mosaic switch for PAM-free and one-pot CRISPR/Cas12a detection.},
journal = {Biosensors & bioelectronics},
volume = {314},
number = {},
pages = {119177},
doi = {10.1016/j.bios.2026.119177},
pmid = {42696818},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a combined with nucleic acid amplification enables highly specific and sensitive detection. However, its broader deployment is constrained by protospacer adjacent motif (PAM) dependence, multistep workflows, and limited reagent practicality. Here, we identify a PAM-independent Cas12a activator, termed mosaic DNA, which exhibits structural features intermediate between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Building on this finding, we develop a PAM-independent mosaic switch triggered by a solid-liquid phase transition, thereby addressing these limitations within a single platform. Distinct from previous reports, our experiments show that this activator is generated prior to the digestion of dsDNA into ssDNA by the lambda exonuclease, forming the basis of lambda exonuclease-driven Cas12a activation, a process we refer to as the mosaic switch. Mosaic switch can detect arbitrary dsDNA with sensitivity comparable to that of PAM-containing dsDNA, and maintain single-nucleotide discrimination. Lyophilizing mosaic switch reagents and encapsulating them in paraffin improve stability and usability, which also enables straightforward one-pot integration with recombinase polymerase amplification (RPA) via a solid-liquid phase transition. Applied directly to 58 extraction-free mpox clinical samples, this platform showed complete concordance (100%) with quantitative PCR. This CRISPR/Cas12a platform maintains analytical performance while broadening the range of targets, simplifying the workflow, and enhancing reagent practicality, showing great potential for clinical deployment.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*DNA/genetics/chemistry
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics/chemistry
DNA, Single-Stranded/chemistry/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42759108,
year = {2026},
author = {Tahira, M and Su, W and Maqsood, FK and Ikram, M and Javaid, MH and Rao, MJ and Zhang, J},
title = {CRISPR/Cas9 engineering: Insights into tomato fruit development, stress resilience via brassinosteroid pathways.},
journal = {Plant physiology and biochemistry : PPB},
volume = {238},
number = {},
pages = {111740},
doi = {10.1016/j.plaphy.2026.111740},
pmid = {42759108},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *Brassinosteroids/metabolism ; *Fruit/growth & development/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Signal Transduction ; *Stress, Physiological ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Tomato (Solanum lycopersicum), a cornerstone of global agriculture and nutrition, has undergone decades of breeding focused on yield and stress resilience. Yet, consumer-driven traits such as fruit uniformity, flavor complexity, and nutritional value remain suboptimal. The emerging convergence of hormonal biology and precision genome editing presents a transformative approach to addressing this gap. Brassinosteroids (BRs), a class of steroidal phytohormones, act as central regulators of cell expansion, tissue patterning, and developmental plasticity. Their signaling cascade, initiated by perception at the BRI1-BAK1 receptor complex and transduced via BES1/BZR1 transcriptional modules, intersects with networks controlling fruit set, morphology, ripening, and stress adaptation. Gene editing through CRISPR/Cas9 technology now enables targeted dissection and manipulation of these BR-regulated nodes with unprecedented accuracy. CRISPR/Cas9 studies have directly characterized BR signaling regulators such as SlBZR1, SlBIN2, and SlBES1, while complementary genetic and transgenic studies have provided functional evidence for other BR-related components, including SlBRI1 and the BR-biosynthetic gene SlDWF4, further supporting the roles of BR signaling in tomato fruit development, ripening, and carotenoid accumulation. Beyond improvement of traits, CRISPR/Cas9 offers the potential to tune BR pathways and their crosstalk with auxin and ethylene, providing a systems-level framework for engineering climate-resilient and nutritionally superior cultivars. This review integrates mechanistic insights into BR signaling with cutting-edge CRISPR/Cas9 applications, positioning tomato as a model for reprogramming fruit development and as a paradigm for next-generation crop improvement.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*Brassinosteroids/metabolism
*Fruit/growth & development/metabolism/genetics
*CRISPR-Cas Systems/genetics
Signal Transduction
*Stress, Physiological
Plant Growth Regulators/metabolism
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
Gene Editing

@article {pmid42786150,
year = {2026},
author = {Ramasamy, M and Li, G and Guo, L and Fang, H and Eid, A and Mandlik, R and Cheng, Y and Niedz, RP and Culver, JN and Coleman, GD and Liu, Z and Mandadi, KK and Qi, Y},
title = {Boosting genome editing in perennial plants by CRISPR-Combo mediated morphogenic gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42786150},
issn = {2041-1723},
support = {IOS-2132693//National Science Foundation (NSF)/ ; IOS-2224203//National Science Foundation (NSF)/ ; 2020-70029- 33161//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2021-70029-36056//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2025-70029-44033//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; },
mesh = {*Gene Editing/methods ; Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Solanum tuberosum/genetics ; Fragaria/genetics/growth & development ; *Genome, Plant ; Populus/genetics ; Plant Roots/genetics ; Transcriptional Activation ; Plant Shoots/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {A major bottleneck in genome editing of many perennial plants is their recalcitrance to transformation and regeneration. To boost genome editing in such perennial crops, transcriptional reprogramming of morphogenic genes is introduced by CRISPR-Combo, a versatile system for simultaneous genome editing and transcriptional activation in plant cells. In potato, we screen 17 morphogenic genes and identify 4 genes (WOX11/12, ARF5, ABI3-1, and ABI3-2) that promote regeneration of genome-edited hairy roots, and 3 of the 4 genes are also found to boost shoot regeneration by Agrobacterium-mediated stable transformation. Similarly, screening of 10 morphogenic genes in citrus leads to the identification of 5 genes (BBM3, FUS3, IPT1, SERK1, and STM) that enhance plant regeneration upon activation. In wild strawberry, we demonstrate that simultaneous activation of Baby Boom genes (BBM1 and BBM2) or of GRF3 and GIF1 reduces the generation time of genome-edited plants by over one month. Moreover, in poplar, we show that simultaneous activation of WUS and WOX11 synergistically promotes plant regeneration without exogenous plant hormones, which leads to a protocol of generating genome-edited poplar shoots in less than one month. Collectively, this study provides efficient strategies for boosting genome editing in four perennial crops.},
}

*Gene Editing/methods
Plants, Genetically Modified/genetics
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Solanum tuberosum/genetics
Fragaria/genetics/growth & development
*Genome, Plant
Populus/genetics
Plant Roots/genetics
Transcriptional Activation
Plant Shoots/genetics/growth & development
Plant Proteins/genetics/metabolism
Genes, Plant

@article {pmid42786170,
year = {2026},
author = {Liu, YV and Suryatenggara, J and Wong, H and Jayasinghe, MK and Tang, JP and Tan, HK and Kwon, J and Zhou, Q and Zhang, J and Moein, S and Ummarino, S and Le, MTN and Doench, JG and Benoukraf, T and Chai, L and Hiwase, D and Thomas, D and Di Ruscio, A and Tenen, DG and Bassal, MA},
title = {Methylation Mesa define functional regulatory elements for targeted gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42786170},
issn = {2041-1723},
support = {P01 HL158688/HL/NHLBI NIH HHS/United States ; 2016-0476//Fondazione Cariplo (Cariplo Foundation)/ ; P01HL158688//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; P01 HL131477/HL/NHLBI NIH HHS/United States ; R00 CA188595/CA/NCI NIH HHS/United States ; R35 CA197697/CA/NCI NIH HHS/United States ; R01 DK136116/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; Animals ; *DNA Methylation/genetics ; Mice ; Promoter Regions, Genetic/genetics ; *Transcriptional Activation ; Epigenesis, Genetic ; CpG Islands/genetics ; Chromatin/metabolism ; CRISPR-Cas Systems ; Epigenome Editing ; 5' Untranslated Regions/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; },
abstract = {DNA methylation regulates transcription, yet the demethylation of canonical elements like promoter CpG islands exhibits inconsistent correlations with gene activation. We hypothesize that causal regulatory elements are defined by biophysical hypersensitivity. Profiling 24 whole-genome bisulfite sequencing samples across diverse human and murine models, we identify Methylation Mesa, narrow (~45-300 bp), structurally conserved epigenetic regulatory elements. Mesa show enrichment in 5' untranslated regions and associate with transcriptional activation significantly better than canonical promoters. To investigate causal regulatory dynamics, we develop CRISPR-DiR, an RNA-based targeted demethylation technology offering greater spatial precision, higher potency, and reduced toxicity compared to CRISPR-TET1. While proximal promoter demethylation initiated limited early transcription, focal demethylation of the Mesa seed acts as the primary driver of exponential CDKN2A (p16) reactivation and robust in vivo tumor suppression. We demonstrate that precise demethylation of a Mesa locus triggers localized demethylation, subsequent activation histone mark deposition, and long-range three-dimensional chromatin rewiring. Thus, Methylation Mesa act as precise, dominant epigenetic regulatory hubs, and CRISPR-DiR as a potent high-resolution tool, establishing a structural framework for biomarker discovery and targeted therapies.},
}

Humans
Animals
*DNA Methylation/genetics
Mice
Promoter Regions, Genetic/genetics
*Transcriptional Activation
Epigenesis, Genetic
CpG Islands/genetics
Chromatin/metabolism
CRISPR-Cas Systems
Epigenome Editing
5' Untranslated Regions/genetics
*Regulatory Sequences, Nucleic Acid/genetics

@article {pmid42786391,
year = {2026},
author = {Ghasemi, R and Heidari, H},
title = {Nucleic Acid-Based Antimicrobial Strategies Against Drug-Resistant Staphylococcus aureus: A Review of Mechanisms of Action and Delivery Approaches.},
journal = {Infectious diseases and therapy},
volume = {},
number = {},
pages = {},
pmid = {42786391},
issn = {2193-8229},
abstract = {Drug-resistant Staphylococcus aureus remains one of the major clinical challenges in both community and hospital settings. Increasing rates of resistance to conventional antibiotics have prompted the development of alternative therapeutic strategies capable of selectively targeting essential bacterial functions. Nucleic acid-based antimicrobial strategies have emerged as promising and precise approaches, enabling the inhibition of gene expression, suppression of virulence, restoration of antibiotic susceptibility, and inhibition of bacterial growth in a sequence-specific manner. This review investigates various studies on antisense oligonucleotides (ASOs), small RNA-based approaches, and CRISPR-Cas systems against S. aureus, with a focus on their antimicrobial efficacy, mechanisms of action, and delivery strategies. These platforms can target genes involved in cell division, transcription, quorum sensing, biofilm formation, and antibiotic resistance. As efficient intracellular delivery remains a major barrier, multiple carriers-including cell-penetrating peptides, nanoparticles, liposomes, DNA nanostructures, and phagemid-based platforms-have been developed to enhance therapeutic efficacy. It has been demonstrated that optimized delivery systems can substantially improve the stability, cellular uptake, and antibacterial activity of nucleic acid therapeutics. Furthermore, the programmability and high target specificity of these agents is a step forward, facilitating the development of precision antimicrobial therapies with the potential for reduced effects on non-target bacterial populations. Overall, the available data support the potential of nucleic acid-based antimicrobials as promising preclinical adjuncts to conventional antibiotics for combating multidrug-resistant S. aureus.},
}

@article {pmid42788230,
year = {2026},
author = {Kumar Mandal, R and Halder, J and Kumar, A and Kurmi, BD and Kaur, K and Sharma, A},
title = {Smart nanocarriers against MDR-tuberculosis: stimuli-responsive strategies for granuloma penetration and next generation therapy.},
journal = {Therapeutic delivery},
volume = {},
number = {},
pages = {1-24},
doi = {10.1080/20415990.2026.2732635},
pmid = {42788230},
issn = {2041-6008},
abstract = {Multidrug-resistant tuberculosis (MDR-TB) remains a global health concern due to long treatment durations, limited drug penetration into granulomatous lesions, systemic toxicity, and the persistence of dormant Mycobacterium tuberculosis. The granuloma microenvironment, characterized by hypoxia, acidic pH, enzymatic activity, dense extracellular matrix, and redox imbalance, acts as a barrier to the effectiveness of conventional antitubercular therapy. Recent advances in nanotechnology have led to the development of stimuli-responsive nanocarriers that enable targeted, controlled, and on-demand drug delivery in response to disease-specific stimuli. This narrative review summarizes peer-reviewed literature published between 2015 and 2025 on stimuli-responsive nanocarrier systems for MDR-TB therapy, retrieved from major scientific databases, including PubMed, Scopus, Google Scholar, ScienceDirect, Web of Science, and ResearchGate, focusing on granuloma-targeted drug delivery and nanomedicine strategies. Stimuli-responsive liposomes, polymeric nanoparticles, dendrimers, and metallic nanocarriers demonstrate enhanced pulmonary accumulation with improved bacterial targeting and reduced systemic toxicity compared to free drugs. In addition, co-delivery strategies incorporating host-directed immunomodulators further improve bactericidal efficacy. Emerging approaches, including CRISPR-Cas‑based nano-therapies, RNA therapeutics, regenerative and stem cell-based strategies, artificial intelligence‑guided nanocarrier design, and biohybrid delivery systems, represent important future directions. Overall, stimuli-responsive nanocarriers, integrated with next-generation technologies, offer a promising pathway toward precise and effective MDR-TB treatment.},
}

@article {pmid42213070,
year = {2026},
author = {Nakanishi, K and Takano, Y and Yamamoto, K and Matsuda, Y and Yano, M and Mito, K and Ichino, T and Tatsumi, K and Li, H and Ohara, K and Munakata, R and Suzuki, H and Sakurai, N and Shibata, D and Osakabe, K and Watanabe, B and Okada, T and Shimomura, K and Takanashi, K and Sugiyama, A and Yazaki, K},
title = {CRISPR/Cas9-mediated genome editing reveals the involvement of a polyphenol oxidase in the shikonin-specific biosynthesis in Lithospermum erythrorhizon.},
journal = {Plant & cell physiology},
volume = {67},
number = {9},
pages = {1557-1572},
doi = {10.1093/pcp/pcag074},
pmid = {42213070},
issn = {1471-9053},
support = {19H05638//Japan Society for the Promotion of Science KAKENHI/ ; 22K05438//Japan Society for the Promotion of Science KAKENHI/ ; 25K08838//Japan Society for the Promotion of Science KAKENHI/ ; JPMJSP2110//Japan Science and Technology Agency (JST) SPRING/ ; NEDO 16100890-0//New Energy and Industrial Technology Development Organization/ ; //RIKEN Cluster for Science, Technology and Innovation Hub/ ; //Institute for the Future of Human Society of Kyoto University for 2023/ ; //Jikei University Research Fund/ ; //Humanosphere Science Research on Sustainable Humanosphere Science/ ; //Kyoto University/ ; },
mesh = {*Lithospermum/genetics/metabolism/enzymology ; *Naphthoquinones/metabolism ; *CRISPR-Cas Systems/genetics ; *Catechol Oxidase/metabolism/genetics ; Plant Roots/metabolism/genetics ; Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; },
abstract = {Shikonin, a 1,4-naphthoquinone derivative produced by some limited Boraginaceae species, exhibits unique pharmacological properties and is also used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production, and the copper-requiring biosynthetic step is shikonin-specific, unlike the byproduct benzoquinones. Despite the long history of shikonin biosynthesis studies for over 40 years, the copper-involved reaction has been still unknown. Here, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Genes encoding PPO, a dicopper-dependent oxidoreductase, was highlighted by the strong synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon (LePPOs), only LePPO1 showed a close correlation with shikonin production. Then, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines, while the content of deoxyshikonofuran, a hydroquinone-type shunt product that branches after 3''-hydroxygeranylhydroquinone in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings address the question of why a copper ion is crucial for shikonin biosynthesis and suggest that LePPO1 participates in naphthalene ring formation. Interestingly, LePPO1 is localized in plastids, whereas shikonin accumulates in the apoplast.},
}

*Lithospermum/genetics/metabolism/enzymology
*Naphthoquinones/metabolism
*CRISPR-Cas Systems/genetics
*Catechol Oxidase/metabolism/genetics
Plant Roots/metabolism/genetics
Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant

@article {pmid42487027,
year = {2026},
author = {Kam, KM and Shiu, TE and Hsieh, CM and Lu, WT and Pan, YY and Lin, CI and Lin, PH and Chuang, YH and Yu, IS and Chen, PL},
title = {Generation and characterization of humanized CD4 knock-in mice expressing chimeric mouse/human CD4 protein.},
journal = {Lab animal},
volume = {55},
number = {10},
pages = {402-412},
pmid = {42487027},
issn = {1548-4475},
support = {112-S0309//National Taiwan University Hospital (NTUH)/ ; },
mesh = {Animals ; Humans ; *Gene Knock-In Techniques ; Mice ; *CD4 Antigens/genetics/metabolism ; Mice, Transgenic/genetics ; CD4-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Humanized mouse models have become indispensable tools for investigating human gene function and disease modeling. However, conventional transgenic approaches carry the risk of unforeseen biological consequences. Here, to address this concern, we developed a novel human CD4 knock-in (hCD4 KI) mouse model using CRISPR-Cas9 gene-editing technology. We replaced the region encoding the first two major extracellular domains of the mouse Cd4 gene, which are critical for interaction with major histocompatibility complex (MHC) class II, with the corresponding human CD4 sequence. Subsequently, we conducted comprehensive physiological and immune system analyses on hCD4 KI mice, including both heterozygous (CD4[m/h]) and homozygous (CD4[h/h]) genotypes. Our investigations revealed a dosage-dependent impact of the hCD4 KI, resulting in a decreased percentage of CD4[+] single-positive cells, accompanied by a corresponding increase in CD8[+] single-positive cells within the thymus. These developmental alterations, evident in the thymus, were also observed in the peripheral lymphatic system such as the spleen and in the peripheral blood, exhibiting an increased population of mature CD8[+] T cells and a decreased proportion of mature CD4[+] T cells. Despite these changes, hCD4 KI mice exhibited normal biological characteristics, including T cell activation and proliferation functions, blood composition, tissue structure and body weight, closely resembling those of wild-type (CD4[m/m]) mice. Our study underscores hCD4 KI mice as a valuable tool for exploring CD4 and MHC class II interactions, with potential for future integration with humanized MHC class II KI mice, offering insights into immune disease mechanisms.},
}

Animals
Humans
*Gene Knock-In Techniques
Mice
*CD4 Antigens/genetics/metabolism
Mice, Transgenic/genetics
CD4-Positive T-Lymphocytes
CRISPR-Cas Systems
Female
Male