@article {pmid42383490,
year = {2026},
author = {Nam, O and Grouneva, I and Mackinder, LCM},
title = {A versatile tool for gene editing in the diatom Thalassiosira pseudonana.},
journal = {Plant physiology},
volume = {202},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag385},
pmid = {42383490},
issn = {1532-2548},
support = {MR/T020679/1//United Kingdom Research and Innovation Future Leaders Fellowship/ ; BB/T017589/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S015337/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R001014/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EP/W024063/1//Engineering and Physical Research Council/ ; },
mesh = {*Diatoms/genetics ; CRISPR-Cas Systems/genetics ; Luminescent Proteins/metabolism/genetics ; Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Diatoms are major contributors to marine primary production, with the centric diatom Thalassiosira pseudonana a powerful model for understanding biogeochemical processes including carbon fixation and silicification. While there are molecular tools available for fluorescent protein (FP) tagging and CRISPR/Cas9 genome editing in T. pseudonana, these require the delivery of multiple vectors or have limited versatility. Additionally, scarless endogenous tagging, that results in a FP fusion expressed from its native genomic location, has yet to be developed. Here, we describe a versatile modular Golden Gate-based toolkit for T. pseudonana that through the delivery of a single-episome via bacterial conjugation enables: (i) FP tagging, (ii) dual FP tagging, (iii) CRISPR/Cas9 genome editing, (iv) simultaneous FP tagging with gene editing, and (v) scarless endogenous FP tagging. We further expand the available parts for T. pseudonana by validating 3 additional FPs and 2 untested promoter/terminator pairs. We demonstrate the versatility of our system by knocking out diatom pyrenoid component 1 (DPC1) while simultaneously GFP tagging the rbcS (Rubisco small subunit), and by endogenously GFP tagging the bestrophin-like protein BST2. While DPC1 knockout does not result in a major pyrenoid structural defect due to unperturbed rbcS-GFP localization to the pyrenoid, we confirm that BST2 localizes to the pyrenoid and exhibits increased fluorescence under low CO2-supporting a role in diatom carbon fixation. Our developed genetic tools provide a robust framework for exploring cellular processes in diatoms, accelerating routine studies and enabling systematic, quantitative and large-scale studies.},
}

*Diatoms/genetics
CRISPR-Cas Systems/genetics
Luminescent Proteins/metabolism/genetics
Green Fluorescent Proteins/genetics/metabolism
*Gene Editing/methods
Plasmids/genetics

@article {pmid42861929,
year = {2026},
author = {Pozzi, B and Lucero, AM and Buggiano, V and Pelaez, MA and García, CC and Srebrow, A},
title = {Targeting CLK1 by CRISPR tools and pharmacological inhibition modulates the innate immune response and dengue virus replication.},
journal = {The Biochemical journal},
volume = {483},
number = {12},
pages = {2307-2325},
doi = {10.1042/BCJ20260367},
pmid = {42861929},
issn = {1470-8728},
support = {Impacto-CONVE-2023-100766162-APN-MCT//Secretaria de Innovacion, Ciencia y Tecnologia (ex Ministerio de Ciencia, Tecnologia e Innovacion) de Argentina/ ; 20020220200041BA//UBA | Secretaría de Ciencia y Técnica, Universidad de Buenos Aires (UBACyT)/ ; PICT 2019-00263//Agencia Nacional de Investigaciones Cientificas y Tecnologicas de Argentina/ ; PICT 2020-00198//Agencia Nacional de Investigaciones Cientificas y Tecnologicas de Argentina/ ; },
mesh = {*Dengue Virus/physiology/drug effects ; *Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors/metabolism ; *Virus Replication/drug effects ; *Immunity, Innate/drug effects/genetics ; Humans ; *Dengue/immunology/genetics ; *CRISPR-Cas Systems ; Animals ; Alternative Splicing ; Interferons/pharmacology ; Protein-Tyrosine Kinases ; },
abstract = {Alternative splicing is a key regulatory mechanism known to be altered upon viral infection. These alterations can arise both from direct viral interference with the splicing machinery and from cellular responses such as activation of innate immunity. Here, we investigated splicing changes shared between dengue virus (DENV) infection and interferon (IFN) treatment in cultured cells, the latter serving as a model for virus-independent innate immune activation. Among the common events, we identified an increased production of non-coding mRNA isoforms from the CLK1 gene, which gives rise to a kinase that phosphorylates splicing factors including SR proteins and spliceosomal components. Consistent with this finding, IFN treatment led to a reduction in CLK1 protein levels. Using stable cell lines with CRISPR/dCas9-mediated modulation of CLK1 expression, we found that silencing CLK1 enhanced the induction of immune response genes, while its overexpression attenuated it. Inhibition of CLK1 kinase activity with the pharmacological inhibitor TG003 further potentiated IFN-induced gene expression and reduced DENV replication. Altogether, these results identify CLK1 as a proviral negative regulator of IFN-stimulated gene expression and suggest that its inhibition could enhance antiviral defenses and become a target for therapeutic strategies.},
}

*Dengue Virus/physiology/drug effects
*Protein Serine-Threonine Kinases/genetics/antagonists & inhibitors/metabolism
*Virus Replication/drug effects
*Immunity, Innate/drug effects/genetics
Humans
*Dengue/immunology/genetics
*CRISPR-Cas Systems
Animals
Alternative Splicing
Interferons/pharmacology
Protein-Tyrosine Kinases

@article {pmid42869019,
year = {2026},
author = {Yuan, S and Yang, S and Huang, Y and Ma, B and Wu, Q and Zhang, X and Yin, T and Chen, L and Zhang, B and Sun, G and He, Y and Duan, H and Hu, L and Hao, S and Cheng, H and Wei, J and Cheng, T},
title = {Single-cell CRISPR screen maps epigenetic regulators of lineage specification and malignancies in human HSPCs.},
journal = {Cell stem cell},
volume = {33},
number = {11},
pages = {1819-1839.e10},
doi = {10.1016/j.stem.2026.09.005},
pmid = {42869019},
issn = {1875-9777},
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/cytology/pathology ; *Cell Lineage/genetics ; *Epigenesis, Genetic ; *Single-Cell Analysis/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Mapping how genetic perturbations influence cell fate is key to understanding human development and disease. Here, we developed a massively parallel single-cell CRISPR (scCRISPR) screening platform in primary human hematopoietic stem and progenitor cells (HSPCs), profiling 322 regulators across 172,382 single-perturbed cells. The resulting multi-layered regulatory framework captured known associations and enabled functional annotation of regulator-phenotype relationships. Specifically, our atlas revealed that regulators acted within perturbation clusters to govern distinct gene programs that shaped cell fate. As proof of concept, we illustrated several examples. SMYD3 emerged as a negative regulator of long-term regenerative capacity. Analysis of lineage output further identified KMT5B, TRIM24, SETD7, and NSD3 as regulators associated with lineage-biased outputs. Finally, integration with clinical datasets identified PRDM10 as a candidate dependency in acute myeloid leukemia (AML). This work establishes a scalable functional genomics platform in a primary human stem cell system.},
}

Humans
*Hematopoietic Stem Cells/metabolism/cytology/pathology
*Cell Lineage/genetics
*Epigenesis, Genetic
*Single-Cell Analysis/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Differentiation/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42880484,
year = {2026},
author = {Lannoo, R and Bechter, O and Daelemans, D},
title = {CRISPR-based interrogation of alternative lengthening of telomeres: mapping dependencies, stress-buffering pathways, and therapeutic vulnerabilities.},
journal = {Cancer letters},
volume = {662},
number = {},
pages = {218888},
doi = {10.1016/j.canlet.2026.218888},
pmid = {42880484},
issn = {1872-7980},
mesh = {Humans ; *Telomere Homeostasis/genetics ; *Telomere/genetics ; *CRISPR-Cas Systems ; *Neoplasms/genetics/therapy/pathology ; DNA Replication ; DNA Repair ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that supports the growth of approximately 5-10% of human tumors and represents an attractive but insufficiently explored therapeutic target. CRISPR-based functional genomics has emerged as a powerful approach to systematically interrogate ALT biology and identify genetic dependencies associated with the ALT state. In this Review, we discuss the CRISPR-based strategies applied to ALT, including hypothesis-driven targeted loss-of-function screens, phenotype-driven optical screens, in silico mining of large-scale dependency datasets, and genome-wide screens in ALT-adjacent states. Across these approaches, recurrent dependencies converge on pathways mediating replication stress buffering, DNA repair, and telomere maintenance, including FANCM, SMARCAL1, and SUB1. Collectively, these findings support a model in which ALT-positive cells rely not on a unique ALT-specific factor, but rather on stress-buffering mechanisms that prevent chronic telomeric dysfunction from triggering replication catastrophe and cell death, highlighting these pathways as promising candidates for further therapeutic investigation.},
}

Humans
*Telomere Homeostasis/genetics
*Telomere/genetics
*CRISPR-Cas Systems
*Neoplasms/genetics/therapy/pathology
DNA Replication
DNA Repair
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42887040,
year = {2026},
author = {Zhang, L and Cai, B and Li, X and Liu, Y and Lu, P and Liu, Y and Lei, W and Mao, G},
title = {Quadruple HLA-I/II gene homo/heterozygous knockout hESC line: generation, multi-omics profiling, pluripotency, and immunogenicity.},
journal = {International immunopharmacology},
volume = {190},
number = {},
pages = {117565},
doi = {10.1016/j.intimp.2026.117565},
pmid = {42887040},
issn = {1878-1705},
mesh = {Humans ; Animals ; *Human Embryonic Stem Cells/immunology/transplantation ; Cell Line ; Mice ; Gene Knockout Techniques ; Multiomics ; CRISPR-Cas Systems ; *Pluripotent Stem Cells ; *Histocompatibility Antigens Class II/genetics ; *Histocompatibility Antigens Class I/genetics ; Teratoma ; Transplantation, Heterologous ; },
abstract = {Human embryonic stem cells (hESCs) exhibit pluripotency, self-renewal, and broad therapeutic potential in diverse disease contexts. Meanwhile, allogeneic grafts derived from hESCs are confronted with the formidable challenge of immune rejection. Specific human leukocyte antigens (HLAs) are critically involved in mediating transplant rejection, thus establishing HLA-deficient stem cells can be an important step toward achieving immune evasion. However, there is currently no consensus on which gene editing strategy can reduce hESC immunogenicity to a level suitable for broad clinical applications. This study performed HLA-A/-B gene hetero/homozygous knockout in an HLA double-knockout hESC line using a CRISPR/Cas9 system, established HLA-DRA[-/-]/-DQA1[-/-]/-A[+/-]/-B[-/-] knockout monoclonal cell lines, and revealed the multi-omics profiles, along with preserved pluripotency and trends to reduced immunogenicity of the hESC lines, through multi-omics analyses, in vitro assessment of pluripotency-related gene expression, in vivo teratoma formation assays and xenotransplantation experiments in mouse models. This study presents two novel monoclonal HLA genes homo/heterozygous knockout hESC lines, along with their multi-omics landscapes, pluripotency and immunogenicity. The findings exhibit crucial application potential and reference value in the development of hypoimmunogenic hESCs for their universal clinical use.},
}

Humans
Animals
*Human Embryonic Stem Cells/immunology/transplantation
Cell Line
Mice
Gene Knockout Techniques
Multiomics
CRISPR-Cas Systems
*Pluripotent Stem Cells
*Histocompatibility Antigens Class II/genetics
*Histocompatibility Antigens Class I/genetics
Teratoma
Transplantation, Heterologous

@article {pmid42955435,
year = {2026},
author = {Suryadevara, L and Srinivas, V and Barathidasan, R and Mhaske, ST and Naik, RR and Gupta, MK and Pramod, RK},
title = {Comparative analysis of single and dual sgRNA CRISPR/Cas9 ribonucleoprotein strategies for efficient Park7 gene editing in primary mouse embryonic fibroblasts.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42955435},
issn = {1573-4978},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Fibroblasts/metabolism ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Protein Deglycase DJ-1/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Parkinson Disease/genetics ; },
abstract = {BACKGROUND: Parkinson's disease (PD) is a progressive neurological condition that predominantly affects dopaminergic neurons in the substantia nigra. Mutations or deletions in the Park7 gene may cause a rare autosomal recessive form of early-onset PD. Generating reliable cellular models with specific Park7 disruption is crucial for elucidating the molecular mechanisms underlying PD pathogenesis.

METHODS AND RESULTS: This study developed a plasmid-free CRISPR/Cas9 genome-editing method for targeting the Park7 gene in primary mouse embryonic fibroblasts (MEFs) using single- and dual-sgRNA-mediated ribonucleoprotein (RNP) system. Gene modification through the single sgRNA strategy was confirmed by the detection of indels by T7 endonuclease assay, whereas gene editing by the dual sgRNA strategy was validated through detection of large deletions by junctional PCR and Sanger sequencing. Deep amplicon sequencing showed an overall editing frequency of 50.68% and 24.87% for single-sgRNA and dual-sgRNA editing strategies, respectively. While single-sgRNA editing predominantly generated small insertions and deletions, large deletions constituted the predominant editing outcome in the dual-sgRNA group, accounting for 22.23% of the total reads. Further, Western blot analysis demonstrated a 12.03 ± 7.6% reduction in PARK7 protein expression following single sgRNA transfection, whereas dual sgRNA transfection showed a significant reduction of 54.89 ± 9.1% (p < 0.0001).

CONCLUSION: This study demonstrates a plasmid-free CRISPR/Cas9 RNP-based approach for targeted Park7 gene editing in primary MEFs. The results underline the importance of considering both the outcomes of genomic editing and the expression of functional protein when evaluating CRISPR/Cas9 approaches. This established approach sets the basis for the development of Park7-edited cellular models to study molecular mechanisms associated with PD and the subsequent development of disease-relevant models.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
Fibroblasts/metabolism
*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*Protein Deglycase DJ-1/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
Parkinson Disease/genetics

@article {pmid42955689,
year = {2027},
author = {Venkat, KR and Kanchanapalli, L and Varadarajan, S},
title = {RNA-RNA Interaction Prediction in Therapeutics and Drug Discovery.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3079},
number = {},
pages = {305-317},
pmid = {42955689},
issn = {1940-6029},
mesh = {Humans ; *Computational Biology/methods ; CRISPR-Cas Systems ; Deep Learning ; *Drug Discovery/methods ; Oligonucleotides, Antisense/therapeutic use/genetics ; Prediction Algorithms ; *RNA/metabolism/genetics/therapeutic use/chemistry ; RNA, Small Interfering/genetics/therapeutic use/metabolism ; },
abstract = {RNA-based therapeutics have emerged as a transformative modality in modern medicine, enabling precise modulation of gene expression and offering new treatment strategies for previously intractable diseases. Central to the success and safety of many RNA therapeutics is a detailed understanding of RNA-RNA interactions (RRIs), including both intended target binding and unintended off-target effects. Computational prediction of RRIs plays a critical role in the design, optimization, and validation of therapeutic RNAs such as antisense oligonucleotides, small interfering RNAs, messenger RNAs, and CRISPR-associated RNAs. This chapter examines the role of RRI prediction in therapeutic and drug discovery pipelines, highlighting computational strategies, deep learning models, and validation frameworks. Examples of applications in oncology, infectious diseases, and genetic disorders illustrate how accurate RRI modeling supports efficacy, safety assessment and contribute to evidence generation for therapeutic development. Challenges and future directions for clinically deployable RRI prediction systems are also discussed.},
}

Humans
*Computational Biology/methods
CRISPR-Cas Systems
Deep Learning
*Drug Discovery/methods
Oligonucleotides, Antisense/therapeutic use/genetics
Prediction Algorithms
*RNA/metabolism/genetics/therapeutic use/chemistry
RNA, Small Interfering/genetics/therapeutic use/metabolism

@article {pmid42956069,
year = {2026},
author = {Parvez, S and Marsh, MC and Owen, SC and Peterson, RT},
title = {A dCas9 proximity reporter for investigating DNA linear and rotational dynamics.},
journal = {PloS one},
volume = {21},
number = {10},
pages = {e0355748},
pmid = {42956069},
issn = {1932-6203},
support = {R00 HG012593/HG/NHGRI NIH HHS/United States ; R01 GM134069/GM/NIGMS NIH HHS/United States ; },
mesh = {*DNA/genetics/chemistry/metabolism ; Humans ; *Genes, Reporter ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Luciferases/genetics/metabolism ; Aptamers, Nucleotide/genetics/metabolism ; },
abstract = {The dCas9 system has rapidly been developed into many tools to explore different aspects of the human genome; the high binding specificity, coupled with the inactive nuclease enzyme, allows for precise recruitment of molecules to specific sequences of DNA. We sought to exploit these capabilities to create a tool for assessing the real-time proximity of two DNA sequences within a biological system. By incorporating aptamers into gRNAs, dCas9 molecules can be used to recruit the β9 or β10 strands of split-NanoLuc® to specific DNA sequences and quantify the proximity of those sequences based on their ability to complex with the luciferase fragment (Δ11S) and produce luminescence. While many tools exist to detect a single DNA sequence, this system is uniquely capable of assessing how two DNA sequences interact with each other. As expected, we found that the interaction of two dCas9 molecules was affected by their linear distance from each other on dsDNA. Surprisingly, we found that their interaction was also strongly influenced by rotational orientation, even for sequences that are close together in linear space. This finding indicates that dCas9 rotational alignment is an important consideration for designing dCas9 systems that target multiple DNA sequences simultaneously. Beyond the findings presented herein, we believe this DNA proximity detection tool has the potential to be adapted for applications involving the proximity and orientation of two DNA sequences.},
}

*DNA/genetics/chemistry/metabolism
Humans
*Genes, Reporter
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Luciferases/genetics/metabolism
Aptamers, Nucleotide/genetics/metabolism

@article {pmid42956939,
year = {2026},
author = {Waqas, M and Nadeem, M and Shahid, MQ and Daowu, H and Shopu, H and Xiongming, D},
title = {Seed Morphogenesis: Genetic Architecture, Regulatory Mechanisms, and Breeding Opportunities.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {113554},
doi = {10.1016/j.plantsci.2026.113554},
pmid = {42956939},
issn = {1873-2259},
abstract = {Seed morphogenesis determines seed size, shape, tissue organization, and developmental patterning, which influence crop yield and quality. This review compiles the genetic architecture, regulatory mechanisms, and breeding strategies for seed morphogenesis in major crops. Functionally validated genes and QTLs are cataloged with clear inclusion criteria to distinguish direct evidence for the causal gene from candidate genes identified by GWAS and linkage mapping. Developmental and environmental signals converge through hormonal crosstalk and epigenetic control, including Polycomb repressive complex 2 (PRC2) mediated histone modifications, DNA methylation, chromatin remodeling, and non-coding RNAs to shape seed morphogenesis. Six central regulatory modules govern seed morphogenesis: endosperm proliferation and cellularization, seed coat development, embryo patterning, hormonal integration, cell cycle and expansion control, and ncRNAs. Conserved and species-specific pathways are compared across Arabidopsis, cereals, and legumes, demonstrating that embryo patterning (LAFL, WOX, HD-ZIP III) is highly conserved, while endosperm development (MINI3/IKU2, AGL62) and seed coat development (SHP1/SHP2, STK, MYB5) are divergent, highlighting the need for species-specific frameworks. Modern technological advances such as high-throughput phenotyping, multi-omics, and CRISPR/Cas9 enable module-level functional validation. Breeding programs can deploy validated module genes through MAS, GS, and genome editing. Despite these advances, phenotyping, G × E interactions, pleiotropy, and the functional validation gap in underutilized crops remain unresolved. These gaps can be overcome by shifting from single-gene level manipulation to network-level engineering, integrating multi-omics and systems biology to translate module-level discoveries into improved seed morphogenesis.},
}

@article {pmid42957655,
year = {2026},
author = {Guckelberger, P and Doughty, BR and Rao, SSP and Munson, G and Tan, Y and Cai, XS and Fulco, CP and Nasser, J and Bergman, DT and Ray, J and Jagoda, E and Gschwind, AR and Sheth, MU and Munger, CJ and Tan, AS and Mualim, KS and Pulido, SG and Mitra, N and Weisz, D and Shamim, MS and Durand, NC and Mahajan, R and Khan, R and Steinmetz, LM and Kanemaki, MT and Lander, ES and Aiden, EL and Engreitz, JM},
title = {Quantitative changes in 3D contact predict changes in enhancer-promoter regulation upon cohesin depletion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42957655},
issn = {2041-1723},
support = {NNF21SA0072102//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; UM1HG011972//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R35HG011324//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; UM1 HG011972/HG/NHGRI NIH HHS/United States ; R01 HL159176/HL/NHLBI NIH HHS/United States ; R01HL159176//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R35 HG011324/HG/NHGRI NIH HHS/United States ; },
mesh = {Cohesins ; *Cell Cycle Proteins/metabolism/genetics ; *Enhancer Elements, Genetic/genetics ; *Promoter Regions, Genetic/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Gene Expression Regulation ; Animals ; Humans ; CRISPR-Cas Systems ; Chromatin/metabolism ; },
abstract = {Enhancers are drivers of gene regulation thought to act via 3D interactions with promoters. However, genome-wide cohesin depletion causes only limited changes in gene expression, despite widespread loss of contact domains, raising questions about how 3D contacts and cohesin influence enhancer function. Here, we introduce CRISPRi of regulatory elements upon degron operation (CRUDO), to quantitatively assess how changes in contact frequency affect enhancer-driven gene regulation. We analyzed 1039 candidate enhancers near five cohesin-dependent genes and identified 30 significant enhancer-gene pairs, 16 with cohesin-dependent effects. While all cohesin-dependent enhancers were distal (>50 Kb), not all distal enhancers were sensitive to cohesin. Instead, changes in enhancer effects correlated with changes in enhancer-promoter 3D contact frequency, which appeared to depend on both genomic distance and other locus-specific factors. Across the genome, most predicted enhancer-gene regulatory interactions do not show substantial contact changes upon cohesin depletion, explaining why only a small subset of genes are cohesin-sensitive. Together, our results illuminate how 3D contacts tune enhancer effects on endogenous gene expression.},
}

Cohesins
*Cell Cycle Proteins/metabolism/genetics
*Enhancer Elements, Genetic/genetics
*Promoter Regions, Genetic/genetics
*Chromosomal Proteins, Non-Histone/metabolism/genetics
*Gene Expression Regulation
Animals
Humans
CRISPR-Cas Systems
Chromatin/metabolism

@article {pmid42957717,
year = {2026},
author = {Wang, HD and Hu, L and Han, J and Cheng, ZH and Yu, HQ and Liu, DF},
title = {Repurposing Cas12a2-mediated abortive infection for efficient and low-toxicity microbial genome editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42957717},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Escherichia coli/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; Shewanella/genetics ; *Genome, Bacterial ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have revolutionized microbial genome engineering, yet counter-selection strategies typically rely on targeted DNA cleavage, where the severe cytotoxicity of double-strand breaks restricts transformation efficiency and clonal recovery. Here, we present an innovative strategy, SuCas12a2-Universal Recombination Editing (SURE), that circumvents these limitations by repurposing the RNA-targeting CRISPR-Cas12a2 system. Instead of directly attacking the genome, SURE utilizes Cas12a2's RNA-triggered collateral activity to induce an abortive infection-like state, providing a potent yet recoverable selective pressure. This strategy increases transformation efficiency by up to 1,000-fold compared to Cas9- or Cas12-based systems while achieving over 90% editing efficiency for gene knockout, insertion, and replacement in Escherichia coli MG1655. We validate the SURE platform's broad utility by extending it to diverse species, including Bacillus subtilis BSZR and Shewanella oneidensis MR-1. Mechanistically, we identify collateral double-stranded DNA degradation as the decisive factor for driving efficient selection. Finally, we apply this system to engineer E. coli MG1655 for efficient assimilation of the plastic monomer ethylene glycol, demonstrating its practical utility. Collectively, this work establishes a universal and robust RNA-guided editing framework that resolves the toxicity-efficiency trade-off inherent to the conventional DNA-targeting methods.},
}

Escherichia coli/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Bacillus subtilis/genetics
Shewanella/genetics
*Genome, Bacterial
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
*Endodeoxyribonucleases/genetics/metabolism

@article {pmid42957818,
year = {2026},
author = {Farahani, MS and Shamsara, M and Moazzeni, SM and Khalili, S and Johnson, B and Darzi, L and Shoae, M and Ghavami, M and Razi, A and Hosseini-Beheshti, E and Moghadam, MF},
title = {T cell engineering by targeted small extracellular vesicles delivering CRISPR/Cas9 system for PD-1 knock-out.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {42957818},
issn = {1432-0614},
mesh = {*CRISPR-Cas Systems ; *T-Lymphocytes/immunology/metabolism ; *Programmed Cell Death 1 Receptor/genetics ; *Extracellular Vesicles/metabolism/genetics ; *Gene Knockout Techniques/methods ; Humans ; *Cell Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Electroporation ; Gene Editing/methods ; Animals ; Plasmids ; },
abstract = {Among the emerging cancer therapies, immunotherapy using T cell engineering stands out as one of the most promising approaches. While the clustered regularly interspaced palindromic repeats (CRISPR/Cas) system is widely recognized as one of the most common gene-editing system tools, the main challenge for in vivo delivery applications remains the lack of an efficient and safe intracellular carrier. Small extracellular vesicles (s-EVs), as natural nanoparticles with low immunogenicity, emerge as a promising candidate for delivering the CRISPR/Cas system. Two guide RNAs (gRNAs) targeting the programmed cell death (PD-1) gene were cloned into the PX-459 vector, and these CRISPR/Cas9 plasmids were loaded into targeted s-EVs by electroporation. Subsequently, T cells were exposed to these s-EVs, and the disruption to the PD-1 gene was examined using flow cytometry and sequencing. Flow cytometry results indicated that newly produced targeted s-EVs exhibited a higher uptake by T cells compared to non-targeted s-EVs. The successful loading of PX-459 plasmids into targeted s-EVs and their functional delivery to T cells were confirmed. Subsequent carboxyfluorescein succinimidyl ester (CFSE) staining and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that the knockout (KO) of the PD-1 gene in T cells significantly enhanced both their cytotoxicity and cytokine release compared to untreated T cells. Targeted s-EVs were used in the current study as a means to deliver the CRISPR/Cas9 system into T cells, enabling genetic modification of target cells. This approach holds the potential to improve and simplify immunotherapy. This study demonstrates a novel approach to genetic modification in T cells using targeted s-EVs containing the CRISPR/Cas9 gene-editing system. KEY POINTS: •CRISPR/Cas9 is a kind of powerful gene editing system in cancer therapeutics. •s-EVs can be applied as a biological carrier for CRISPR/Cas9 system. • T cell engineering-based immunotherapy is a promising form of anticancer therapy.},
}

*CRISPR-Cas Systems
*T-Lymphocytes/immunology/metabolism
*Programmed Cell Death 1 Receptor/genetics
*Extracellular Vesicles/metabolism/genetics
*Gene Knockout Techniques/methods
Humans
*Cell Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Electroporation
Gene Editing/methods
Animals
Plasmids

@article {pmid42957850,
year = {2026},
author = {Roux, S and Neri, U and Bushnell, B and Fremin, BJ and George, NA and Gophna, U and Hug, LA and Camargo, AP and Wu, D and Ivanova, N and Kyrpides, NC and Eloe-Fadrosh, EA},
title = {Planetary-scale metagenomic analysis reveals CRISPR targeting dynamics across ecosystems.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42957850},
issn = {2058-5276},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; ERC-AdG 787514//European Commission (EC)/ ; 2016-03686//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (Conseil de Recherches en Sciences Naturelles et en Génie du Canada)/ ; },
abstract = {Interactions between microorganisms and their mobile genetic elements (MGEs), including viruses and plasmids, impact microbiome dynamics. CRISPR-Cas systems are important regulators of these host-MGE interactions, but a global understanding of the diversity, activity and roles of CRISPR spacers across Earth's biomes is still lacking. Here we develop a computational approach to search short-read data and collect 794 million CRISPR spacers across 367,609 public metagenomes, from which we identified 1.18 billion hits between 41 million spacers and 2.5 million viruses and plasmids. While spacers were highly variable across samples, those matching known viruses were more prevalent and conserved, consistent with a positive selection pressure associated with phage targeting. Among these, however, we find evidence suggestive of recurrent entry of phage genomes into non-host microbial cells, further blurring the line between CRISPR targeting and virus-host interactions. These results highlight key drivers of CRISPR targeting patterns likely to influence strain-level diversification and selection processes in microbiomes.},
}

@article {pmid42118102,
year = {2026},
author = {Pan, Y and Nie, X and Lü, P},
title = {A Highly Sensitive ERA-CRISPR/Cas13a Assay for Detection of Vibrio vulnificus.},
journal = {Journal of AOAC International},
volume = {109},
number = {6},
pages = {1034-1041},
doi = {10.1093/jaoacint/qsag038},
pmid = {42118102},
issn = {1944-7922},
support = {//Postdoctoral Funding Project of Weihai City/ ; NY2023007//Key Technology Research and Development (Modern Agriculture) Program of Zhenjiang City/ ; },
mesh = {*Vibrio vulnificus/isolation & purification/genetics ; Humans ; Animals ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Ostreidae/microbiology ; Food Microbiology ; Seafood/microbiology ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Vibrio vulnificus is a halophilic marine bacterium widely distributed in coastal environments and seafood products and is responsible for severe human infections and foodborne disease outbreaks. Rapid and sensitive detection of V. vulnificus is therefore critical for clinical diagnosis and seafood safety monitoring.

OBJECTIVE: The objective of this study was to develop and evaluate a rapid, sensitive, and specific nucleic acid detection method for V. vulnificus suitable for application in complex biological and food matrices.

METHODS: An enzymatic recombinase amplification (ERA)-clustered regularly interspaced short panlindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a)-based nucleic acid detection assay was established by coupling ERA with Cas13a-mediated collateral cleavage of fluorescent RNA reporters. The analytical performance of the assay was systematically evaluated using V. vulnificus standard plasmids, artificially contaminated human serum samples, and spiked oyster homogenates.

RESULTS: The assay exhibited high specificity for V. vulnificus, with no cross-reactivity observed for other common Vibrio species. The LOD was 1 × 10-1 copies/μL for standard plasmid templates. In complex matrices, including human serum and oyster samples, the assay achieved an LOD of 1 × 101 CFU/mL. The entire detection process was completed under isothermal conditions within 45 min.

CONCLUSION: A sensitive and specific ERA-CRISPR/Cas13a-based assay for the detection of V. vulnificus was successfully developed and validated. The method demonstrates strong analytical performance in both biological and seafood matrices.

HIGHLIGHTS: A rapid isothermal ERA-CRISPR/Cas13a assay for V. vulnificus detection was developed. High specificity was achieved without cross-reactivity to other Vibrio species. The assay enables sensitive detection in serum and oyster samples. The method shows potential for routine seafood safety surveillance and marine pathogen monitoring.},
}

*Vibrio vulnificus/isolation & purification/genetics
Humans
Animals
Limit of Detection
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Ostreidae/microbiology
Food Microbiology
Seafood/microbiology
Sensitivity and Specificity

@article {pmid42135078,
year = {2026},
author = {Guo, Y and Zhu, Z and Zhu, C and Shang, B and Huang, Y and Xu, W and Yang, L},
title = {Exponential signal amplification through coupled rolling circle transcription and CRISPR/Cas13a cleavage for ultrasensitive molecular diagnostics.},
journal = {Trends in biotechnology},
volume = {44},
number = {11},
pages = {3361-3391},
doi = {10.1016/j.tibtech.2026.04.018},
pmid = {42135078},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Transcription, Genetic ; SARS-CoV-2/genetics/isolation & purification ; DNA-Directed RNA Polymerases ; *COVID-19/diagnosis/virology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Rapid Diagnostic Tests ; },
abstract = {Rapid, sensitive nucleic acid detection is crucial for clinical diagnostics, food safety, and environmental monitoring. We present the Sensitive Padlock Rolling-circle INtegrated Transcription with clustered regularly interspaced short palindromic repeats (CRISPR)/LwCas13a detector (SPRINT-C), a transformative one-pot isothermal platform capable of detecting single molecules within 10 min. By rationally designing padlock probes, we enable seamless coupling of three enzymatic reactions: template-mediated padlock ligation, rolling circle transcription (RCT), and CRISPR/LwCas13a cleavage. Our mechanistic studies reveal that the substantially higher transcription rate of T7 RNA polymerase (5.97 cycles/s) relative to LwCas13a cis-cleavage (<0.019 s[-1]) results in incomplete cis-cleavage of RCT products, generating short RNA fragments that re-initiate the amplification cascade. This creates exponential amplification, transforming linear RCT into a self-perpetuating chain reaction. SPRINT-C achieves ultrasensitivity (1-2 copies/reaction) with single-nucleotide specificity, successfully detecting severe acute respiratory syndrome coronavirus 2 variants in clinical samples, including 'gray-zone' specimens (Ct 37-40). Integration with lateral flow enables an equipment-free visual detection method, demonstrating versatility across pathogen detection, genetically modified organism identification, and microRNA analysis.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
Transcription, Genetic
SARS-CoV-2/genetics/isolation & purification
DNA-Directed RNA Polymerases
*COVID-19/diagnosis/virology
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Rapid Diagnostic Tests

@article {pmid42212547,
year = {2026},
author = {Schreuder, A and de Lint, K and Mỹ Phúc Võ, H and Góis, MM and Kampen, RA and San Martin Alonso, M and Nootenboom, I and Garzero, V and Wendel, TJ and Wolthuis, RMF and Noordermeer, SM},
title = {Finding novel vulnerabilities of hypomorphic BRCA1 alleles.},
journal = {Molecular oncology},
volume = {20},
number = {11},
pages = {2820-2838},
pmid = {42212547},
issn = {1878-0261},
support = {Sylvie M Noordermeer VI.Vidi.192.039//Exacte en Natuurwetenschappen/ ; //Oncode Institute/ ; },
mesh = {Humans ; *BRCA1 Protein/genetics/metabolism ; *Alleles ; Mutation, Missense ; Genomic Instability ; Cell Line, Tumor ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {With the recent rise in CRISPR/Cas9-mediated genome-wide synthetic lethality screens, many new synthetic lethal targets have been identified for diseases with underlying genetic causes such as tumours with BRCA1 mutations. Such screens often use full deficiency of a protein to identify novel vulnerabilities. However, patient-derived mutations not only result in full loss of the protein but often also concern missense mutations with hypomorphic phenotypes. Here, we study the genetic vulnerabilities of two previously described hypomorphic BRCA1 missense mutations and compare these to a BRCA1-depleted setting to study whether this affects screening for synthetic lethal interactions. Our research showed that BRCA1[I26A] mutated cells have very similar vulnerabilities to BRCA1 wild-type cells, confirming its low tumorigenic effect. In contrast, the BRCA1[R1699Q] mutation induced a more similar phenotype to BRCA1-deficient cells. For this mutation, we also unveiled a unique vulnerability to the loss of NDE1. Specifically in BRCA1[R1699Q] mutated cells, and not BRCA1-proficient or -deficient cells, NDE1 loss leads to increased genomic instability. Altogether, our findings highlight the importance to differentiate between patient-derived mutations when assessing novel treatment targets.},
}

Humans
*BRCA1 Protein/genetics/metabolism
*Alleles
Mutation, Missense
Genomic Instability
Cell Line, Tumor
CRISPR-Cas Systems
Phenotype

@article {pmid42441958,
year = {2026},
author = {Wang, J and Nava Cruz, C and Walsh, JD and desRanleau, E and Nikonorova, IA and Barr, MM},
title = {A C. elegans model for functional analysis of conserved ADPKD variants in cilia, extracellular vesicles, and sensory signaling.},
journal = {Genetics},
volume = {234},
number = {3},
pages = {},
pmid = {42441958},
issn = {1943-2631},
support = {DK059418//National Institutes of Health (NIH)/ ; DK116606//National Institutes of Health (NIH)/ ; #959686//Polycystic Kidney Disease Foundation Award/ ; #1443775//Polycystic Kidney Disease Foundation Award/ ; //CGC/ ; P40OD010440//National Institutes of Health-Office of Research Infrastructure Programs/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Cilia/metabolism/genetics ; *TRPP Cation Channels/genetics/metabolism ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Polycystic Kidney, Autosomal Dominant/genetics/metabolism/pathology ; Disease Models, Animal ; Humans ; Signal Transduction ; CRISPR-Cas Systems ; },
abstract = {Interpreting the pathogenic significance of missense variants in human disease gene candidates remains a major challenge in precision medicine. Autosomal dominant polycystic kidney disease (ADPKD), a common cause of kidney failure, results from mutations in PKD1 or PKD2, encoding polycystin-1 and polycystin-2. Here, we establish C. elegans as a platform for the functional classification of PC2 variants by characterizing PKD-2C180S, the C. elegans ortholog of the likely pathogenic human variant PC2C331S. Using CRISPR/Cas9 genome editing with dual-color fluorescent reporters and super-resolution imaging, we show that PKD-2C180S reduces cell-body protein levels, abolishes ciliary and extracellular vesicle (EV) localization, and eliminates sensory function, phenocopying a pkd-2 null allele. In heterozygous animals, PKD-2C180S is recessive and exerts no dominant-negative effect on wild-type PKD-2 trafficking, protein levels, or function, establishing that PKD-2 is haplosufficient in this model. PKD-2C180S also abolishes ciliary and EV localization of the PC1 homolog LOV-1 and reduces LOV-1 cell-body levels, phenocopying pkd-2 null animals, consistent with PC2 acting as a molecular chaperone for PC1 stability and trafficking. Genetic epistasis analysis shows that PKD-2C180S protein levels and ciliary trafficking defects are intrinsic to the mutant protein and independent of lov-1 and the ciliary kinesin-3 klp-6. Quantitative analysis reveals that LOV-1•PKD-2 is more abundant at the ciliary membrane and more efficiently packaged into EVs than PKD-2 alone in lov-1 mutants. We conclude that PC2C331S may perturb protein stability and/or polycystin complex formation prior to ciliary/EV trafficking. This work establishes a C. elegans pipeline for mechanistic classification of conserved ADPKD-associated missense variants.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
*Cilia/metabolism/genetics
*TRPP Cation Channels/genetics/metabolism
*Caenorhabditis elegans Proteins/genetics/metabolism
*Polycystic Kidney, Autosomal Dominant/genetics/metabolism/pathology
Disease Models, Animal
Humans
Signal Transduction
CRISPR-Cas Systems

@article {pmid42619262,
year = {2026},
author = {Kubek-Luck, K and Velazquez, J and Yao, X and Gabriels, J and Hsu, C and Arora, S and Bontadelli, L and Gilbart, Y and Aihara, F and Li, H and Bouadjemi, F and Ooi, J and Nunes, J and Sigoillot, F and Weiler, J and Meseck, E and Mansfield, K and Hudry, E},
title = {Leveraging host-cell modulators of adeno-associated vector transduction to tailor viral biodistribution.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6329-6345},
doi = {10.1016/j.ymthe.2026.08.024},
pmid = {42619262},
issn = {1525-0024},
mesh = {*Dependovirus/genetics ; Animals ; Humans ; *Genetic Vectors/genetics/administration & dosage ; *Transduction, Genetic ; Mice ; Cell Line ; Liver/metabolism ; Genetic Therapy/methods ; CRISPR-Cas Systems ; Tissue Distribution ; },
abstract = {Clinical successes in gene therapy using adeno-associated vectors (AAVs) are offering a hopeful path toward the correction of several monogenic disorders. At its core, AAV gene delivery relies on multiple interactions between the capsid or vector genome and numerous host-cell factors, a complex process that remains incompletely understood. Whether different serotypes similarly hijack the intracellular machinery in a cell, and whether these processes are necessarily conserved across species, are not known. To identify host factors enabling or preventing transduction across species or serotypes, CRISPR-Cas9 whole-genome knockdown screens were conducted in mouse (AML12) and human (HuH-7) cell lines before transduction with AAV2 or AAV9. Key common entry factors (AAVR, GPR108) were confirmed, with many other serotype- and/or species-specific host-cell modulators identified. Interestingly, while serotype-specific differences were observed for successful transduction of a specific cell line, species-specific divergences were even more striking when comparing results across murine and human cells for the same serotype. Leveraging these data, we developed a novel approach to detarget liver transduction by transiently downregulating the expression of key entry factors in this tissue using GalNac-small interfering RNAs (siRNAs) prior to AAV9 administration. Such manipulation not only blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.},
}

*Dependovirus/genetics
Animals
Humans
*Genetic Vectors/genetics/administration & dosage
*Transduction, Genetic
Mice
Cell Line
Liver/metabolism
Genetic Therapy/methods
CRISPR-Cas Systems
Tissue Distribution

@article {pmid42675859,
year = {2026},
author = {Taifour, S and Wong, CW and Wallis, C and Wang, E and Tie, AWJ and Woodward, E and Waryah, C and Hosseinpour, M and Lam, D and Lister, R and Dymond, L and Woo, A and Houghton, PJ and Iyer, KS and Norret, M and Evans, CW and Winteringham, L and Gaudieri, S and Blancafort, P},
title = {Targeting of the oncogenic fusion EWSR1-FLI1 in Ewing sarcoma by CRISPR/dCas9 silencers.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6456-6484},
doi = {10.1016/j.ymthe.2026.08.042},
pmid = {42675859},
issn = {1525-0024},
mesh = {*Sarcoma, Ewing/genetics/therapy/pathology ; Humans ; *Oncogene Proteins, Fusion/genetics ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Mice ; *Proto-Oncogene Protein c-fli-1/genetics ; *RNA-Binding Protein EWS/genetics ; Xenograft Model Antitumor Assays ; Gene Silencing ; },
abstract = {Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle to clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR-nuclease-dead Cas9 (dCas9) systems adapted for epigenetic repression (dCas9-Krüppel-associated box [KRAB]) to silence oncogenic drivers with high selectivity. As proof of principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable, non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. Collectively, we characterize an effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.},
}

*Sarcoma, Ewing/genetics/therapy/pathology
Humans
*Oncogene Proteins, Fusion/genetics
Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Mice
*Proto-Oncogene Protein c-fli-1/genetics
*RNA-Binding Protein EWS/genetics
Xenograft Model Antitumor Assays
Gene Silencing

@article {pmid42675860,
year = {2026},
author = {Liu, J and Wang, S and Wang, B and Yu, T and Liu, X and Li, M and Ku, MC and Wang, R and Chai, R and Zheng, WV and Wang, Y},
title = {Rational and computation-assisted engineering of a compact and efficient CRISPR-Cas12f genome editor.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6485-6500},
doi = {10.1016/j.ymthe.2026.08.043},
pmid = {42675860},
issn = {1525-0024},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; Protein Engineering ; },
abstract = {The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. Single guide RNA (sgRNA) scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
*CRISPR-Associated Proteins/genetics/metabolism
Mutation
Protein Engineering

@article {pmid42687383,
year = {2026},
author = {Sun, Y and Li, L and Wang, X and Han, L and Zhao, Y and Tan, Y and Feng, Y and Qi, W and Ma, H},
title = {Optimization of AsCas12f1-mediated long-term gene repression.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6501-6512},
doi = {10.1016/j.ymthe.2026.09.001},
pmid = {42687383},
issn = {1525-0024},
mesh = {*Gene Silencing ; DNA Methylation ; Epigenome Editing ; *CRISPR-Cas Systems ; Humans ; Epigenesis, Genetic ; Histones/metabolism/genetics ; *Gene Editing/methods ; },
abstract = {A compact and programmable epigenome editor can be utilized for long-term gene silencing, making it a powerful tool for biological research and biomedical applications. Here, we present an enhanced CRISPR-based epigenome editor based on deactivated Acidibacillus sulfuroxidans Cas12f1 (dAsCas12f1) variants, which incorporate epigenetic modulators for histone methylation and DNA methylation. First, we compared the gene silencing achieved by various dCas12f1 variants fused with Krüppel-associated box (KRAB). The optimized variant dCas12f1-V8.1-KRAB, named AsCas12f1-based miniaturized CRISPRi (AminiCRi), demonstrated high-efficacy gene silencing comparable to that of dSpCas9-KRAB. To establish a miniature Cas-based system for long-term silencing, we generated dCas12f1-V8.1 fused with KRAB and DNA methyltransferases (Dnmt3A/3L), named AsCas12f1-based miniaturized CRISPRoff (AminiCRoff). AminiCRoff enables simultaneous deposition of repressive histone H3K9 trimethylation marks and DNA methylation at target loci. Transient delivery of AminiCRoff allows stable suppression of H2B gene expression to 30% of baseline levels for up to 50 days, which is more durable than dSpCas9-based CRISPRoff. The compact size of the system is potentially packed into a single adeno-associated virus (AAV), which is frequently used for gene therapy. Here, we developed a highly efficient miniature Cas-based epigenetic editor for long-term gene silencing, which provides a good foundation for epigenetic therapy of genetic or chronic diseases with abnormal gene expression.},
}

*Gene Silencing
DNA Methylation
Epigenome Editing
*CRISPR-Cas Systems
Humans
Epigenesis, Genetic
Histones/metabolism/genetics
*Gene Editing/methods

@article {pmid42760781,
year = {2026},
author = {DeWitt, M and Campo-Fernandez, B and Roy, S and Kleinhenz, ALW and Davila, A and Lennon, M and Vu, JT and Krishnappa, N and Said, S and Tseng, CH and Park, SH and Bao, G and Magis, W and Martin, DIK and Boffelli, D and Wyman, SK and Kohn, DB and Walters, MC and Romero, Z},
title = {Development of CRISPR_SCD001, an autologous hematopoietic stem cell gene therapy for sickle cell disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6260-6274},
doi = {10.1016/j.ymthe.2026.09.020},
pmid = {42760781},
issn = {1525-0024},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Animals ; Humans ; Mice ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism ; *Hematopoietic Stem Cell Transplantation/methods ; Gene Editing ; *CRISPR-Cas Systems ; Hemoglobin, Sickle/genetics ; Gene Therapy Agents ; Transplantation, Autologous ; Antigens, CD34/metabolism ; Disease Models, Animal ; },
abstract = {We developed CRISPR_SCD001, an autologous cellular therapy in which the pathogenic HBB sickle allele is corrected by CRISPR-Cas9-mediated homology-directed repair, using a single-stranded oligodeoxyribonucleotide as DNA donor template. The drug product (DP) from plerixafor-mobilized peripheral blood CD34[+] cells from sickle cell disease patients and healthy donors were used for (1) pharmacodynamic assessments to interrogate therapeutic potential, (2) evaluation of genotoxicity, and (3) in vivo characterization by a non-GLP toxicology study in NBSGW mice. Large-scale DP lots yielded 89% ± 5% CD34[+] purity and had a viability of 84% ± 6%. Mean gene correction at HBB was 22% ± 4% of alleles, with 51% ± 6% of allelic disruption. Edited erythroid cultures produced 39% ± 5% adult hemoglobin and 40% ± 6% fetal hemoglobin, reducing sickle hemoglobin from 89% to 22% ± 6%. In NOD,B6.SCID Il2rg [-/-] Kit[W41/W41] (NBSGW) mice, CRISPR_SCD001 engrafted robustly (59% ± 25%), maintained editing frequencies comparable to injected cells (∼20% gene correction), and showed polyclonal distribution of the edited cells; no noteworthy treatment-related toxicity or pathologic evidence was found by a blinded histopathology assessment. Our protocol generates a clinical-grade, cryopreserved CD34[+] cell product that corrects the sickle mutation, restores anti-sickling hemoglobins, and meets pre-clinical safety criteria, establishing a reproducible manufacturing process suitable for clinical translation. These data support initiation of a first-in-human phase 1 trial of CRISPR_SCD001 for sickle cell disease patients presenting with severe pathology.},
}

*Anemia, Sickle Cell/therapy/genetics
Animals
Humans
Mice
*Genetic Therapy/methods
*Hematopoietic Stem Cells/metabolism
*Hematopoietic Stem Cell Transplantation/methods
Gene Editing
*CRISPR-Cas Systems
Hemoglobin, Sickle/genetics
Gene Therapy Agents
Transplantation, Autologous
Antigens, CD34/metabolism
Disease Models, Animal

@article {pmid42768749,
year = {2026},
author = {Vacca, R and Della Volpe, L and Gualandi, N and Shiroshita, K and Carsana, E and Giannetti, K and Gaddoni, C and Benedicenti, F and Gega, B and Arévalo, S and Zarghamian, P and Beretta, S and Castiello, MC and Brombin, C and Canarutto, D and Merelli, I and Cathomen, T and Montini, E and Villa, A and Naldini, L and Di Micco, R},
title = {p38 MAPK inhibition safeguards genome integrity during gene editing of hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {11},
pages = {6234-6242},
doi = {10.1016/j.ymthe.2026.09.024},
pmid = {42768749},
issn = {1525-0024},
mesh = {*Hematopoietic Stem Cells/metabolism/drug effects/cytology ; *p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism ; *Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems ; Mice ; *Protein Kinase Inhibitors/pharmacology ; },
abstract = {Ex vivo genome editing of hematopoietic stem and progenitor cells (HSPCs) holds significant therapeutic potential but remains constrained by genotoxic risks associated with nuclease-induced DNA double-strand breaks, DNA donor template delivery and sensing, and proliferation-induced stress during ex vivo manipulation. These processes can lead to chromosomal instability, large on-target deletions, donor mis-integration, off-target events, and impaired long-term stem cell function, raising safety concerns for clinical translation. Here, we evaluate the impact of transient p38 MAPK inhibition on genomic integrity during clinically relevant CRISPR-Cas9 editing and show that this intervention attenuates ex vivo culture-associated stress without increasing detectable genotoxic outcomes. Comprehensive genotoxicity analyses, including quantification of large on-target deletions, adeno-associated viral vector mis-integration, and CAST-seq mapping of translocations, reveal no measurable differences in gene editing-associated structural alterations upon p38 MAPK inhibition, while micronuclei were significantly reduced. Importantly, long-term xenotransplantation followed by whole-exome sequencing shows that p38i-treated HSPCs display a reduced mutational burden without evidence of increased genomic alterations. Collectively, these findings identify transient p38 MAPK inhibition as a strategy to improve the quality and long-term fitness of gene-edited HSPCs without detectable adverse effects on the genome editing outcomes, supporting its further evaluation as a refinement to clinically relevant CRISPR-Cas9 editing workflows.},
}

*Hematopoietic Stem Cells/metabolism/drug effects/cytology
*p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/metabolism
*Gene Editing/methods
Humans
Animals
CRISPR-Cas Systems
Mice
*Protein Kinase Inhibitors/pharmacology

@article {pmid42788767,
year = {2026},
author = {Li, J and Gong, L and Huang, X and Liu, F and Mei, J and Fu, S and Qiu, L and Zhang, D and Cheng, D and Chen, X},
title = {Field-deployable visual detection of Burkholderia gladioli and its toxigenic pathovar using a one-step RPA-CRISPR/Cas13a assay.},
journal = {Microbiology spectrum},
volume = {14},
number = {11},
pages = {e0214326},
pmid = {42788767},
issn = {2165-0497},
support = {2024KF22//Open Research Project of Zhejiang Key Laboratory of Public Health Detection and Pathogenesis Research/ ; 2025JK141, 2025JKY0458//Zhejiang Science and Technology Plan for Disease Prevention and Control/ ; LMRY26H200007//Mindray Joint Fund of Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Burkholderia gladioli/genetics/isolation & purification/metabolism/classification ; *CRISPR-Cas Systems/genetics ; Bongkrekic Acid/metabolism ; Food Microbiology ; Rapid Diagnostic Tests ; },
abstract = {Burkholderia gladioli pathovar cocovenenans produces the potent mitochondrial toxin bongkrekic acid (BA), causing severe foodborne outbreaks. Conventional detection methods are constrained by prolonged turnaround times and the need for sophisticated instruments, limiting their utility for on-site surveillance. In this study, two independent one-step RPA-CRISPR/Cas13a detection systems were established: one targeting a species-level marker for broad screening of B. gladioli and the other targeting the bonA gene cluster for specific identification of the toxigenic pathovar. The complete one-step reaction was performed at 37°C for 30 min, and results were visually interpreted using a portable blue-light transilluminator. The visual detection limit reached 1 × 10[0] copy/μL for both assays, with no cross-reactivity observed against a panel of six non-target strains. Component validation experiments confirmed that all listed constituents were indispensable for fluorescence signal generation. The practical applicability of the platform was evaluated using 100 naturally contaminated black fungus and mushroom substrate samples. The species-level assay returned positive rates of 78% (39/50) for black fungus and 42% (21/50) for substrate samples, while the bonA-targeted assay yielded positive rates of 38% (19/50) and 8% (4/50), respectively. The visual readout results were in 100% concordance with conventional quantitative real-time PCR. This field-deployable, equipment-free visual detection platform holds considerable promise for on-site surveillance and early warning of BA-producing B. gladioli in food and environmental matrices.IMPORTANCEBongkrekic acid (BA) poisoning caused by Burkholderia gladioli pv. cocovenenans is a lethal food safety threat, particularly in China and Southeast Asia. Current detection methods depend heavily on laboratory-based instruments, limiting their utility for on-site surveillance. This study developed two independent one-step RPA-CRISPR/Cas13a detection systems: one for broad screening of B. gladioli at the species level and the other for specific identification of the toxigenic pathovar. The one-step reaction was completed at 37°C within 30 min, and results were visually interpreted using only a portable blue-light transilluminator. The visual detection limit reached 1 × 10[0] copy/μL, and the method exhibited 100% concordance with quantitative real-time PCR when applied to 100 naturally contaminated samples. This field-deployable, equipment-free platform provides a practical tool for on-site surveillance and early warning of BA-producing B. gladioli in food and environmental matrices.},
}

*Burkholderia gladioli/genetics/isolation & purification/metabolism/classification
*CRISPR-Cas Systems/genetics
Bongkrekic Acid/metabolism
Food Microbiology
Rapid Diagnostic Tests

@article {pmid42953289,
year = {2026},
author = {Cheng, X and Ruan, W and Jin, Z and Suo, L and Ma, Y and Jiang, L and Zheng, W and Li, N and Hu, W},
title = {Reshaping the scissors: From directed evolution to AI-driven design for enhanced Cas performance.},
journal = {Biodesign research},
volume = {8},
number = {4},
pages = {100124},
pmid = {42953289},
issn = {2693-1257},
abstract = {CRISPR/Cas gene editing holds great promise for applications in agricultural breeding and gene therapy; however, native Cas proteins face bottlenecks such as off-target effects, PAM limitations, and delivery challenges. This review explores a three-tiered, progressive, and synergistic analytical framework comprising directed evolution, rational structural design, and integrated protein engineering. Following the logic of "random exploration-precision modification-system integration," it systematically clarifies the principles, advantages, disadvantages, and complementary interactions among these three optimization techniques. By comprehensively mapping the entire technical pathway for Cas protein improvement, this study fills a gap in systematic analysis within the field and provides a theoretical framework for tiered, synergistic modification to support the development of high-performance editors. The paper further analyzes a novel research and development paradigm in which AI and protein language models profoundly empower Cas modification. It summarizes intelligent toolchains such as structure prediction, virtual mutation, and de novo design of artificial Cas proteins, and highlights the unique advantages of AI in overcoming the performance trade-offs inherent in traditional protein engineering. At the same time, it identifies existing challenges, such as data scarcity and insufficient model interpretability, and looks ahead to how multimodal gene-editing large language models and automated closed-loop experimental systems will drive the iterative development of miniaturized, broad-spectrum, and highly precise editing tools, thereby facilitating the clinical and industrial implementation of gene-editing technologies.},
}

@article {pmid42953311,
year = {2026},
author = {Xia, K and Wang, X and Wu, Z and Chu, X},
title = {Molecular Technologies for Intracellular mRNA Imaging and Their Recent Biological Applications.},
journal = {Chemical & biomedical imaging},
volume = {4},
number = {10},
pages = {2138-2158},
pmid = {42953311},
issn = {2832-3637},
abstract = {Understanding the spatial and temporal behaviors of individual mRNA molecules is essential for elucidating gene expression regulation and cellular function. Imaging these processes within cells offers unique insights into transcriptional heterogeneity, mRNA transport, translation, and degradation dynamics. In this Review, we summarized the principal molecular technologies for intracellular mRNA imaging and tracking. These approaches are classified based on their compatibility with fixed cells versus live cells. Fixed-cell FISH-based methods provide spatial resolution and multiplex quantification. Live-cell systems, such as RNA stem-loop binding proteins, CRISPR/Cas platforms, fluorogenic RNA aptamers, fluorescent chemical labeling strategies, molecular beacons, and DNA circuits, enable real-time visualization of both endogenous and exogenous mRNA transcripts in live cells. We finally discuss the challenges and potential future directions for intracellular mRNA imaging. We anticipate that this Review will inspire the development of the next generation of mRNA imaging technologies in diverse biological applications.},
}

@article {pmid42953345,
year = {2026},
author = {Chen, H and Xiao, J and Diao, B and Zheng, S and Shao, H and Liu, L},
title = {Recent advances in bacterial detection techniques targeting genetic, protein, and enzymatic biomarkers.},
journal = {Journal of pharmaceutical analysis},
volume = {16},
number = {10},
pages = {101584},
pmid = {42953345},
issn = {2214-0883},
abstract = {Rapid and accurate diagnosis of bacterial infections is essential for effective clinical management, food safety, and environmental monitoring. Traditional culture-based methods are time-consuming and inadequate for addressing urgent challenges such as antimicrobial resistance (AMR). This review categorizes recent bacterial detection strategies into three major biomarker types: genetic, protein, and enzymatic. For each category, we outline the underlying detection mechanisms, evaluate analytical performance, and highlight representative technologies, including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) assays, advanced immunoassays, aptamer sensors, and enzyme-based assays. Meanwhile, we provide a detailed description of representative examples for each representative technology. Key metrics such as sensitivity, detection time, portability, and point-of-care (POC) applicability are compared to reveal the strengths and limitations of each approach. Translational progress is discussed with emphasis on commercialized platforms, field-deployable devices, and multimodal workflows. Finally, major technical bottlenecks are identified, and future directions are proposed for developing robust, rapid, and widely accessible bacterial diagnostics.},
}

@article {pmid42953358,
year = {2026},
author = {Liu, HB and Kumar, T and Abu-Ellail, FFB and Lin, XQ and Li, CJ and Xu, CH and Arif, M and Khan, W and Ismail, M and Zhao, LP and Sakr, EAE and Haq, NU and Lu, X},
title = {CRISPR/Cas genome editing strategies to enhance abiotic stress tolerance in sugar crops.},
journal = {Frontiers in genome editing},
volume = {8},
number = {},
pages = {1942811},
pmid = {42953358},
issn = {2673-3439},
abstract = {Sugar crops including sugarcane, sugar beets, and sweet sorghum, are fundamental for global sucrose supply and crucial feedstocks for bioethanol production and industrial bioproducts. However, yield and sucrose accumulation are significantly influenced by abiotic stresses, including drought, salinity/alkalinity, heat, chilling/freezing, and heavy metal toxicity. The polygenic complexity of stress tolerance, particularly in sugarcane with its large, polyploid, and haplotype-diverse genome, presents significant challenges for conventional breeding, rendering it slow and imprecise. This review combines advances in genome-editing technologies, including CRISPR/Cas systems (Cas9 and Cas12a), along with transcriptional techniques (CRISPRa/i), base editors, and prime editors. These tools, combined with the progress in reference genomes, haplotype-resolved assemblies, and pangenomes, have facilitated precise gene discovery, genomic selection, and targeted editing. CRISPR-based approaches offer promising avenues for enhancing key stress-responsive pathways governing oxidative homeostasis, membrane integrity, osmotic regulation, hormone signaling, and ion-homeostasis in sugar crops. Prominent candidate targets include transcription factors (TF), signaling components, raffinose biosynthesis genes implicated in freezing tolerance, regulators of salt and alkaline stress, and determinants of heavy metal tolerance and sequestration. Translational challenges, such as off-target effects, low transformation efficiencies, and editing multiple homologues or haplotypes, are being addressed such as DNA-free ribonucleoprotein (RNP) delivery, high-throughput screening, haplotype-specific target design, and haplotype-aware guide RNA engineering based on unique genetic variation sites across different gene haplotypes. Collectively, the integration of advanced CRISPR toolkits with enhanced genomic information resources and phenotyping capabilities holds significant potential for accelerating the development of precision-bred, multi-stress-resilient sugar crops that sustain yield, quality, and industrial value under abiotic stress conditions.},
}

@article {pmid42953961,
year = {2026},
author = {Martinson, JNV and Song, LCT and Rubin, BE},
title = {DNA defense systems become a fitness liability during plasmid conjugation.},
journal = {Nucleic acids research},
volume = {54},
number = {20},
pages = {},
pmid = {42953961},
issn = {1362-4962},
support = {//Lyda Hill Philanthropies/ ; //Acton Family Giving/ ; //Valhalla Foundation/ ; //Hastings/Quillin Fund/ ; //Silicon Valley Community Foundation/ ; //CH Foundation/ ; //Laura and Gary Lauder and Family/ ; //Sea Grape Foundation/ ; //Emerson Collective/ ; //Mike Schroepfer and Erin Hoffman Family Fund/ ; G-2302-06692//Anne Wojcicki Foundation/ ; //Leona M. and Harry B. Helmsley Charitable Trust/ ; //m-CAFEs (Microbial Community Analysis & Functional Evaluation in Soils)/ ; //Lawrence Berkeley National Laboratory/ ; DE-AC02-05CH11231//U.S. Department of Energy/ ; //Shurl and Kay Curci Foundation/ ; //Innovative Genomics Institute/ ; },
mesh = {*Escherichia coli/genetics ; *Plasmids/genetics ; *Conjugation, Genetic ; *Gene Transfer, Horizontal ; *Genetic Fitness ; Type IV Secretion Systems/genetics/metabolism ; DNA Transposable Elements ; CRISPR-Cas Systems ; Extrachromosomal DNA ; },
abstract = {Horizontal gene transfer via conjugative plasmids is a major driver of bacterial evolution. While antibiotic exposure selects for the resistance genes carried by some plasmids, much uncertainty remains about how plasmids persist and spread in microbial communities in the absence of such external selection. Here, we show that conjugative plasmid transfer selectively reduces the fitness of non-permissive recipients through the process of lethal zygosis: a phenomenon in which repeated Type IV Secretion System (T4SS) engagement damages recipients that fail to establish the plasmid. We found that this contact-dependent antagonism effectively clears a niche for plasmid donors by turning recipients' DNA defenses into a liability. Blocking establishment of the plasmid RK2 with CRISPR-Cas or restriction-modification caused strong fitness defects in Escherichia coli recipients by preventing expression of plasmid-encoded exclusion genes and leaving cells exposed to repeated T4SS engagement. We leveraged this insight to resolve an obstacle in bacterial editing by developing transposon vectors that co-deliver exclusion genes, increasing the efficiency of gene editing by orders of magnitude. Our results reveal the role of "coercive assimilation" in horizontal gene transfer, exposing a tradeoff where the advantage of bacterial immunity to foreign DNA is counterbalanced by increased susceptibility to lethal zygosis.},
}

*Escherichia coli/genetics
*Plasmids/genetics
*Conjugation, Genetic
*Gene Transfer, Horizontal
*Genetic Fitness
Type IV Secretion Systems/genetics/metabolism
DNA Transposable Elements
CRISPR-Cas Systems
Extrachromosomal DNA

@article {pmid42955098,
year = {2026},
author = {Lu, T and Liu, X and Wang, C and Huang, B and Mo, Z and Guo, J and Gong, X and Zhu, Y and Tong, X and Hu, CY and Wang, C},
title = {Mdfi overexpression promotes oxidative muscle fiber formation without affecting muscle development in mature skeletal muscles.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {42955098},
issn = {1420-9071},
support = {2022B0202090002//Guangdong Provincial Key R&D Program/ ; 2023KJ106//Provincial Agricultural Science Innovation and Promotion Project/ ; 2023GZ26//Project of State Key Laboratory of Swine and Poultry Breeding Industry/ ; },
mesh = {Animals ; *Muscle Fibers, Skeletal/metabolism/cytology ; Mice ; *Muscle Development/genetics ; *Muscle, Skeletal/metabolism ; Oxidative Phosphorylation ; Mice, Transgenic ; Calcium Signaling ; Tamoxifen/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Skeletal muscles are composed of multinucleated fibers with distinct metabolic and contractile properties. Skeletal muscles support locomotion and systemic metabolism, and their dysfunction underlies the pathogenesis of muscular dystrophies. The MyoD family inhibitor (Mdfi) has been implicated in muscle biology, yet its function in muscle development and muscle fiber type conversion remains largely unclear. Here, we developed the first tamoxifen (TMX)-inducible, skeletal muscle-specific Mdfi overexpression mouse model using CRISPR/Cas9 and Cre[ERT2] integration, enabling spatiotemporal control of Mdfi activity to mimic therapeutic modulation. This model revealed that Mdfi exerts dual-phase regulatory roles: suppressing early postnatal myogenesis while driving glycolytic-to-oxidative fiber switching in mature muscle. Mechanistically, elevated Mdfi activates the calcium signaling pathway to enhance mitochondrial oxidative phosphorylation, directly linking its function to improved muscle fatigue resistance. These Mdfi overexpression-induced phenotypic alterations exhibit stable transgenerational inheritance patterns. Our findings establish Mdfi as a key regulator of muscle plasticity and metabolic adaptation, providing a genetically tractable model for investigating muscle development and energy metabolism. Importantly, this study opens new avenues for understanding and potentially modulating fiber-type composition in muscular dystrophies and other disorders involving metabolic insufficiency, offering a conceptual framework for future therapeutic interventions aimed at improving muscle health.},
}

Animals
*Muscle Fibers, Skeletal/metabolism/cytology
Mice
*Muscle Development/genetics
*Muscle, Skeletal/metabolism
Oxidative Phosphorylation
Mice, Transgenic
Calcium Signaling
Tamoxifen/pharmacology
CRISPR-Cas Systems

@article {pmid42955124,
year = {2026},
author = {Huang, W and Cui, J and Wen, Z and Wu, Y and Shan, X and Sun, D and Han, F and Zhang, X},
title = {CauYL, encoding a magnesium chelatase H subunit, is required for chlorophyll accumulation and normal leaf coloration in cauliflower.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {11},
pages = {},
pmid = {42955124},
issn = {1432-2242},
support = {32472753//the National Natural Science Foundation of China/ ; 32472753//the National Natural Science Foundation of China/ ; CAAS-ASTIP-IVFCAAS//the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; 24JCJQJC00160//Tianjin Natural Science Foundation Project/ ; 2022YFF1003000//the National Key Research and Development Program of China/ ; 25ZXZSSS00320//Tianjin Science and Technology Program Project/ ; },
mesh = {*Lyases/genetics/metabolism ; *Plant Leaves/genetics/metabolism ; *Chlorophyll/metabolism ; Phenotype ; *Pigmentation/genetics ; *Brassica/genetics/enzymology ; Chromosome Mapping ; Photosynthesis/genetics ; Mutation ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Fine mapping and CRISPR/Cas9 validation identified CauYL(BOB03G201420), encoding the H subunit of magnesium chelatase, as the causal gene for the yellow-green leaf phenotype in cauliflower. As the primary photosynthetic pigment in plants, chlorophyll captures light energy to drive photosynthesis and regulate plant growth. In addition, it confers green coloration to plants by absorbing blue and red light wavelengths. Despite the important role of chlorophyll in vegetables, the genetic regulation of chlorophyll accumulation in cauliflower remains largely unknown. In this study, we identified a cauliflower mutant "K-6" from an ethyl methanesulfonate (EMS) mutagenized population, that displayed a stable yellow-green phenotype. The mutant showed much lower chlorophyll contents and decreased photosynthetic rate compared with the WT plants. Genetic inheritance analysis revealed that this yellow-green trait was controlled by a recessive gene. The target gene, designated CauYL, was mapped to an approximately 103.07 kb region on chromosome 3, based on BSA-seq and traditional linkage analyses. We identified a candidate gene BOB03G201420 encoding the H subunit of magnesium chelatase. Sequence analysis revealed a non-synonymous single nucleotide polymorphism (SNP) within the coding sequence, resulting in an alanine-to-threonine substitution at a highly conserved residue. We employed CRISPR/Cas9 to knockout the CauYL gene in the WT background, and successfully generated CauYL mutants, which showed a yellow-green trait resembling that of the K-6 mutant. Transcriptome analysis showed that differentially expressed genes between K-6 and WT were significantly enriched in photosynthesis, riboflavin metabolism, and related pathways. Collectively, our results demonstrate that the CauYL gene is essential for maintaining the green coloration and photosynthetic competence of cauliflower, providing a valuable genetic target for improving photosynthetic efficiency and leaf color traits in cauliflower breeding.},
}

*Lyases/genetics/metabolism
*Plant Leaves/genetics/metabolism
*Chlorophyll/metabolism
Phenotype
*Pigmentation/genetics
*Brassica/genetics/enzymology
Chromosome Mapping
Photosynthesis/genetics
Mutation
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42310715,
year = {2026},
author = {Naik Bukke, SP and Prajapati, S and Thalluri, C and Yadav, S and Singh, AP and Gupta, N},
title = {Exosome engineering and molecular tools for targeted therapy of brain-infecting pathogens: delivery systems, signaling pathways, and therapeutic applications.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42310715},
issn = {1477-3155},
mesh = {*Exosomes/metabolism/chemistry ; Humans ; Animals ; Signal Transduction ; Blood-Brain Barrier/metabolism ; *Brain/microbiology/metabolism ; *Drug Delivery Systems/methods ; CRISPR-Cas Systems ; },
abstract = {Brain infections, caused by various pathogens (such as viruses, bacteria, fungi, or parasites), have proven challenging to treat due to limited drug diffusion through the blood-brain barrier and the presence of intracellular reservoirs. As biologically derived nanocarriers, exosomes have emerged as viable candidates for crossing physiological barriers and effectively delivering target molecules into the central nervous system. This review aims to summarize what is currently known about exosome biogenesis, cargo sorting, and immunological function in relation to infectious disease. In addition, it provides information on how different pathogens have taken advantage of exosomal pathways to increase their virulence and modulate the immune response, while also suggesting options for the therapeutic engineering of exosomes. It critically evaluates technological advances made in exosome engineering, such as CRISPR/Cas9-based cargo loading, ligand-directed surface modification of exosomes, targeted delivery of nucleic acids, and creation of stimuli-responsive release systems for exosome cargo for their potential application as precision therapies against pathogens that infect the brain. Pharmacokinetic data and biodistribution studies, along with studies examining how route of administration, inflammatory status, and receptor mediated uptake affect CNS targeting efficacy reflect that exosome engineering offers a novel platform for creating precision therapeutics against pathogens that infect the brain.},
}

*Exosomes/metabolism/chemistry
Humans
Animals
Signal Transduction
Blood-Brain Barrier/metabolism
*Brain/microbiology/metabolism
*Drug Delivery Systems/methods
CRISPR-Cas Systems

@article {pmid42439777,
year = {2026},
author = {Peng, B and Tominaga, M and Wang, C and Speight, RE and Ishii, J},
title = {Transposons as Tools for Future Genome Engineering in Yeasts and Filamentous Fungi.},
journal = {Yeast (Chichester, England)},
volume = {43},
number = {6},
pages = {276-289},
pmid = {42439777},
issn = {1097-0061},
support = {JPJS00420230009//Japan Society for the Promotion of Science/ ; },
mesh = {*Fungi/genetics ; *DNA Transposable Elements/genetics ; *Genome, Fungal ; *Genetic Engineering/methods ; Metabolic Engineering/methods ; *Yeasts/genetics ; CRISPR-Cas Systems ; },
abstract = {Transposons are fundamental genetic elements that have profoundly shaped the architecture of eukaryotic genomes. Yeasts and filamentous fungi have emerged as important chassis organisms for bioingredient production in synthetic biology and metabolic engineering. In this review, we summarise the current understanding and future opportunities in the development of transposon-based tools for genome engineering in these fungal systems. Fungal inverted terminal repeat (ITR) DNA transposons, as well as long terminal repeat (LTR) and non-LTR retrotransposons, can accelerate genomic mutagenesis, facilitating the screening of superior genotypes and phenotypes. CRISPR-associated transposons (CASTs) hold considerable potential for site-specific integration of large transgenes, bypassing the limitations imposed by low homologous recombination (HR) efficiency in non-Saccharomyces hosts. Overall, transposon-based tools represent a valuable and underexplored avenue to accelerate genome engineering and strain development in yeasts and filamentous fungi.},
}

*Fungi/genetics
*DNA Transposable Elements/genetics
*Genome, Fungal
*Genetic Engineering/methods
Metabolic Engineering/methods
*Yeasts/genetics
CRISPR-Cas Systems

@article {pmid42579301,
year = {2026},
author = {Li, Z and Qiao, J and Zhang, Q and Liu, P and Zheng, C and Zhou, Q and Ji, M and Zhu, Z and Li, X and Zhang, H},
title = {Dual-line Genome-scale CRISPR Screening Enables Robust Target Gene Discovery.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {61},
pages = {},
pmid = {42579301},
issn = {2198-3844},
support = {32220103009//National Natural Science Foundation of China/ ; CARS-24//China Agriculture Research System of MOF and MARA/ ; //Hubei Hongshan Laboratory/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Tephritidae/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genomics/methods ; *Gene Editing/methods ; },
abstract = {Pooled CRISPR screening enables genome-scale functional gene discovery by linking genetic perturbations to cellular phenotypes. However, its application in insects remains limited, as systematic differences often obscure the universal functional signals and compromise the reliable identification of genetic regulators. We found that Cas9 genomic integration was associated with distinct transcriptional states, editing activity, and screening outcomes. To address this limitation, we developed a dual-line CRISPR screening framework that prioritizes genes with concordant effects across independently derived Cas9-expressing insect cell lines. In Bactrocera dorsalis, dual-line consensus screening increased in vivo validation of candidate genes from 60% in single-line screens to 90%. Under cold stress, this framework identified candidate regulators of cold tolerance, including a previously uncharacterized gene whose perturbation altered embryonic survival at low temperature. These results show that dual-line screening can filter cell line-specific artifacts and improve functional genomic screening for reliable genetic target discovery in insects.},
}

Animals
*CRISPR-Cas Systems/genetics
*Tephritidae/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Genomics/methods
*Gene Editing/methods

@article {pmid42826482,
year = {2027},
author = {Zhang, J and Huang, H and Xu, J and Zhu, Z and Wang, C and Miao, P and Wang, H and Li, M},
title = {CRISPR-based point-of-care platform for EBV DNA screening and monitoring in nasopharyngeal cancer plasma.},
journal = {Talanta},
volume = {313},
number = {Pt A},
pages = {130685},
doi = {10.1016/j.talanta.2026.130685},
pmid = {42826482},
issn = {1873-3573},
mesh = {Humans ; *Herpesvirus 4, Human/genetics/isolation & purification ; *DNA, Viral/blood/genetics ; *Point-of-Care Systems ; *Nasopharyngeal Neoplasms/virology/diagnosis/blood ; *Point-of-Care Testing ; CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/virology/diagnosis/blood ; *Epstein-Barr Virus Infections/diagnosis/blood ; Rapid Diagnostic Tests ; },
abstract = {Plasma circulating cell-free Epstein-Barr virus (EBV) DNA is a clinically validated biomarker for early nasopharyngeal carcinoma (NPC) detection and longitudinal disease monitoring. However, current quantitative PCR (qPCR) assays require sophisticated instrumentation and specialized laboratory infrastructure, constraining their accessibility in resource-limited settings. Here, we present two point-of-care testing platforms integrating the RPA-CRISPR/Cas13a system and oil-phase magnetic bead transfer (OMT) method for POCT detection: dμCRISPR, a droplet microfluidic chip for digital quantification, and idCRISPR, an integrated lateral flow device for visual screening. The OMT method achieves efficient and stable EBV DNA extraction from plasma within 8 min. The dμCRISPR platform provides absolute quantitative results within 60 min with an analytical sensitivity of 1 copy/μL. In parallel testing of 24 clinical plasma samples, dμCRISPR were consistent with qPCR. Additionally, the idCRISPR platform enables naked-eye screening within 50 min with a detection limit of 10 copies/μL, showing results consistent with qPCR in the 30 clinical samples evaluated. Together, these platforms form a dual-track strategy for precise quantification and rapid screening, supporting the end-to-end management of NPC across diverse healthcare environments.},
}

Humans
*Herpesvirus 4, Human/genetics/isolation & purification
*DNA, Viral/blood/genetics
*Point-of-Care Systems
*Nasopharyngeal Neoplasms/virology/diagnosis/blood
*Point-of-Care Testing
CRISPR-Cas Systems
*Nasopharyngeal Carcinoma/virology/diagnosis/blood
*Epstein-Barr Virus Infections/diagnosis/blood
Rapid Diagnostic Tests

@article {pmid42892015,
year = {2027},
author = {Guo, H and Jiang, Y and Li, D and He, K and Chen, Y and Li, Y and Dai, S and He, Y},
title = {Rapid and visual detection of group B Streptococcus based on an ERA-CasMNPs colorimetric biosensor.},
journal = {Talanta},
volume = {313},
number = {Pt A},
pages = {130794},
doi = {10.1016/j.talanta.2026.130794},
pmid = {42892015},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *Streptococcus agalactiae/isolation & purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; Sensitivity and Specificity ; Recombinases/metabolism ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; },
abstract = {Group B Streptococcus (GBS) is a major pathogen responsible for adverse perinatal outcomes; rapid and accurate detection is crucial for preventing maternal-neonatal transmission and for guiding clinical interventions. Current GBS detection methods are limited by long assay times, insufficient sensitivity, and high equipment and operational requirements, restricting their application in resource-limited settings and rapid detection scenarios. Targeting the cfb gene of GBS, this study developed ERA-CasMNPs by integrating enzymatic recombinase amplification (ERA), CRISPR/Cas12a-mediated specific recognition and trans-cleavage activity of ssDNA, and horseradish peroxidase (HRP)-modified magnetic nanoparticle complexes (MNPs)-based colorimetric signal output. The ERA-CasMNPs biosensor enables rapid and sensitive visual detection of GBS within 85 min, with an LOD95 of 23.3 copies/μL (95% confidence interval: 15.6-34.8 copies/μL). Moreover, evaluation using 135 cultured clinical bacterial isolates demonstrated that ERA-CasMNPs achieved a sensitivity of 97.33%, a specificity of 98.33%, and a detection accuracy of 97.78%, comparable to qPCR. These results indicate that the ERA-CasMNPs biosensor provides a sensitive and visual method for rapid detection of GBS and supports its potential for further evaluation in resource-limited and rapid-testing settings.},
}

*Biosensing Techniques/methods
*Colorimetry/methods
*Streptococcus agalactiae/isolation & purification/genetics
Humans
*Streptococcal Infections/diagnosis/microbiology
Sensitivity and Specificity
Recombinases/metabolism
CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests

@article {pmid42926546,
year = {2026},
author = {Valentin-Alvarado, LE and Yoon, PH and Lopez, SC and Shulgina, Y and Langeberg, C and Armbruster, E and Ur-Rehman, MM and Loi, K and Taveneau, C and Esain-Garcia, I and Koka, M and Zhang, ZT and Kashin, D and Schwede, F and Burgio, G and O'Connell, MR and Doherty, EE and Cate, JHD and Knott, GJ and Doudna, JA},
title = {tRNA-derived second messengers confer bacterial immunity.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42926546},
issn = {2692-8205},
abstract = {Bacterial small-molecule messengers that function in diverse immune signaling pathways have been thought to derive strictly from biosynthesis using nucleotide and cofactor substrates. Here we report a nuclease-driven immune signaling pathway in which tRNA cleavage generates a specific small molecule that triggers cell death in response to RNA-guided transcript recognition in prokaryotes. We identify a class of type VI-B CRISPR systems in which the RNA-guided nuclease Cas13 and its accessory transmembrane protein, 2TMβ, are co-encoded in an operon and are jointly required for bacteriophage defense. Upon target RNA recognition, Cas13 activates 2TMβ by producing mono- and dinucleotide messengers bearing 2',3'-cyclic uridine phosphate ends by tRNA cleavage. Activated 2TMβ destroys infected cells by forming a multimeric complex that disrupts cell membranes to mediate cell death. These findings demonstrate RNA-programmed tRNA cleavage for second-messenger biogenesis, revealing a new logic for prokaryotic immune signaling.},
}

@article {pmid42951305,
year = {2026},
author = {Deshwal, A and Tripathi, RM},
title = {Nanozyme-Driven Next-Generation miRNA Biosensing.},
journal = {Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology},
volume = {18},
number = {6},
pages = {},
doi = {10.1002/wnan.70083},
pmid = {42951305},
issn = {1939-0041},
support = {TAR/2022/000074//Anusandhan National Research Foundation (ANRF)/ ; },
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques ; Humans ; Animals ; *Nanostructures/chemistry ; *Nanotechnology ; },
abstract = {MicroRNAs (miRNAs) are small, noncoding RNA molecules nearly 22 nucleotides in length that contribute significantly to regulating gene expression. Their aberrant expression patterns are associated with disease progression, including cardiovascular and neurological disorders and cancer. However, the rapid detection of miRNAs remains challenging due to their low abundance, sequence similarity, and degradation. Conventional methods of detecting miRNAs, such as Northern blotting, microarray analysis, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and next-generation sequencing (NGS), offer significant benefits for miRNA profiling, though they have drawbacks, including limited specificity, complex workflows, relatively high cost, and specialized equipment requirements. Advanced methodologies driven by nanotechnology, especially those that combine clustered regularly interspaced short palindromic repeats (CRISPR)/Cas approaches, provide revolutionary developments. Nanozymes enhance the stability and biocompatibility of catalytic amplification, while CRISPR technologies enable real-time, multiplex, and ultrasensitive miRNA detection with accurate sequence recognition. The rapidly expanding field encompasses diverse nanozyme architectures, recognition mechanisms, amplification strategies, and signal-transduction platforms, providing a broad basis for critically assessing advances, limitations, and emerging opportunities in nanozyme-enabled miRNA biosensing. Nanozyme-enabled miRNA detection has evolved across diverse sensing platforms, such as colorimetric and optical assays, sophisticated photoelectrochemical (PEC) and electrochemiluminescence (ECL) biosensors, and portable and paper-based diagnostic technologies. The convergence of nanozyme catalysis, CRISPR-based recognition, and advanced sensing strategies holds promise for sensitive, selective, and portable miRNA diagnostics.},
}

*MicroRNAs/analysis
*Biosensing Techniques
Humans
Animals
*Nanostructures/chemistry
*Nanotechnology

@article {pmid42951549,
year = {2026},
author = {Jiang, W and Zhu, Y and Kang, J and Zhu, Z and Qian, K and Ji, H and Wang, Q and Qin, Y and Wu, L},
title = {CRISPR/Cas12a-based simplified lateral flow assay for sensitive detection of OTA and AFB1.},
journal = {Analytica chimica acta},
volume = {1425},
number = {},
pages = {346312},
doi = {10.1016/j.aca.2026.346312},
pmid = {42951549},
issn = {1873-4324},
mesh = {*Aflatoxin B1/analysis ; *Ochratoxins/analysis ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Rapid Diagnostic Tests ; *Biosensing Techniques/methods ; Food Contamination/analysis ; *Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {Mycotoxin contamination in agricultural commodities poses serious health risks, necessitating sensitive and user-friendly detection methods for food safety monitoring. Here, we present a multiplex CRISPR/Cas12a-assisted lateral flow assay (MC-LFA) for the parallel detection of Ochratoxin A (OTA) and Aflatoxin B1 (AFB1). The platform employs a duplex Switch design in which target binding releases an activator sequence (Probe 2), triggering Cas12a trans-cleavage of a half-blocking reporter (HBR). Cleavage generates dsDNA products labeled with Dig/FAM (for OTA) or TAMRA/FAM (for AFB1), which are captured at distinct test lines via specific antibody recognition. An integrated streptavidin (SA) capture line binds intact HBR through biotin-streptavidin interactions, effectively replacing conventional magnetic separation steps. The total assay time is approximately 65 min, including a 45-min target identification and a 15-min Cas12a reaction followed by a 5-min lateral flow readout. This approach achieves visual detection limits of 50 pg/mL for OTA and 250 pg/mL for AFB1. The assay exhibits excellent linear correlation over broad concentration ranges and demonstrates high accuracy in double-blind testing (97.22% for OTA and 94.44% for AFB1). Notably, the platform requires only aptamer replacement for different targets while maintaining the same Cas12a and crRNA sequences, substantially reducing experimental costs. Furthermore, by co-localizing detection probes in a single tube, an "OR" logic gate strategy enables parallel screening for both mycotoxins with a single test line. This cost-effective, sensitive, and operationally simple platform possesses strong potential for on-site food safety monitoring in resource-limited settings.},
}

*Aflatoxin B1/analysis
*Ochratoxins/analysis
*CRISPR-Cas Systems/genetics
Limit of Detection
Rapid Diagnostic Tests
*Biosensing Techniques/methods
Food Contamination/analysis
*Endodeoxyribonucleases/metabolism/genetics

@article {pmid42952969,
year = {2026},
author = {Chetapalli, C and Karuppaiah, ND and Garlapati, D and Veeraragavan, S and Sreeharsha, RV and Valsalan, VN and Gopal, D and Ramakrishnan, B},
title = {Recent biotechnological advances and technology readiness level of natural products derived from marine fungi.},
journal = {3 Biotech},
volume = {16},
number = {11},
pages = {481},
pmid = {42952969},
issn = {2190-572X},
abstract = {Bioprospecting involves the systematic exploration of natural resources to identify bioactive secondary metabolites with potential for development into commercially valuable products. In this review, more than a hundred bioactive compounds derived from marine fungi were analysed, with emphasis on their chemical diversity, biological activities, commercialisation status, economic relevance, and Technology Readiness Levels (TRLs). The compounds represented diverse metabolite classes, including polyketides, alkaloids, terpenoids, peptides, azaphilones, and other bioactive secondary metabolites with pharmaceutical, nutraceutical, cosmeceutical, industrial, and environmental applications. Among the reviewed compounds, only approximately 10-20 have been marketed, while the majority remain at early stages of development. TRL assessment showed that most marine fungal-derived products are concentrated at TRL 1-3, a smaller number of compounds and bioprocesses have progressed to TRL 4-5, and highly commercialised products reach TRL 9. In India, pharmaceutical applications involving anticancer, antimicrobial, and antiviral compounds were assessed at TRL 3-5, nutraceuticals at TRL 5-7, cosmeceuticals at TRL 6-8, and industrial enzymes at TRL 7-9. The main barriers to commercialisation were low and unstable metabolite yields, structural complexity, regulatory and bioprospecting constraints, inadequate translational investment and industrial infrastructure, and high financial and technical risks during late-stage development. Integrated approaches including strain improvement, fermentation optimization, chemoenzymatic synthesis, OSMAC, in-situ product removal, genome mining, metabolic engineering, Chemi-genomic approaches, and CRISPR/Cas-based pathway engineering offer opportunities to improve production and increase TRLs. Overall, the findings demonstrate a substantial discovery-to-commercialization gap in marine fungal biotechnology. Advancing strain development, scalable bioprocessing, safety and regulatory validation, synthetic biology, industrial partnerships, and translational infrastructure is essential to convert promising low-TRL discoveries into commercially viable products and strengthen the contribution of marine fungi to a sustainable circular bioeconomy.},
}

@article {pmid41656573,
year = {2026},
author = {Diao, HP and Meng, HX and Xu, XJ and Zhang, ZL and Zhang, JF and Guo, YF and Hwang, I and Song, SJ},
title = {Knocking Out Two Polyphenol Oxidase Genes Significantly Improves Recombinant Protein Purification in Nicotiana benthamiana.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {5588-5596},
pmid = {41656573},
issn = {1467-7652},
support = {2024200CB0110//Beijing Life Science Academy/ ; ZR2024QC068//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Nicotiana/genetics/metabolism/enzymology ; *Catechol Oxidase/genetics/metabolism ; *Recombinant Proteins/isolation & purification/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {Efficient purification remains a key technical challenge affecting the recovery efficiency of recombinant proteins in plant-based systems. Complex metabolites, particularly polyphenols, often cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from Nicotiana benthamiana, as responsible for these effects. Using CRISPR-Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of virus surface proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol-protein interactions, minimised aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.},
}

*Nicotiana/genetics/metabolism/enzymology
*Catechol Oxidase/genetics/metabolism
*Recombinant Proteins/isolation & purification/metabolism/genetics
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid42136068,
year = {2026},
author = {Giroux, B and LeBreux, K and Feyzeau, L and Goulet, MC and Goulet, C and Michaud, D},
title = {Gene Editing of Nicotiana benthamiana Architecture for Space-Efficient Production of Recombinant Proteins in Closed Environments.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {5600-5615},
pmid = {42136068},
issn = {1467-7652},
support = {//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Nicotiana/genetics/metabolism/growth & development ; *Gene Editing/methods ; *Recombinant Proteins/biosynthesis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Lactones/metabolism ; Dioxygenases/genetics/metabolism ; },
abstract = {Indoor vertical farming (VF) offers practical advantages for the cultivation of plant protein bio-factories including plant uniformity, product consistency, water/nutrient recycling and production cycles on a year-round basis. Much progress has been achieved toward the development of innovative systems for artificial lighting, automated irrigation, plant handling, environmental control and space use optimization in VF systems. Here, we used a CRISPR/Cas9 gene editing approach to generate mutant lines of protein expression host Nicotiana benthamiana presenting a compact, space-efficient phenotype suited to VF systems. Our strategy consisted of altering apical dominance by suppressing the synthesis of strigolactone, a negative regulator of axillary bud outgrowth. Strigolactone-depleted lines were generated by hindering the expression of either Carotenoid cleavage dioxygenase 7 (CCD7) or Carotenoid cleavage dioxygenase 8 (CCD8), two key enzymes of the strigolactone synthetic pathway. Knocking out of either enzyme had no impact on the plant's growth rate but drastically influenced its auxin/cytokinin ratio, leaf proteome and overall architecture. The ΔCCD mutants exhibited an axillary growth-oriented development pattern, altered glycolytic and malate-processing metabolic fluxes to drive pyruvate and cytokinin production, and a spatial footprint reduced by 45%-50% compared to the LAB strain commonly used for protein expression. Most importantly, recombinant protein yields per plant were maintained in the mutant lines, as illustrated with GFP and rituximab, a chimeric antibody of therapeutic value. Our data confirm the usefulness of ΔCCD7 and ΔCCD8 knockouts leading to strigolactone depletion for the generation of compact, space-efficient N. benthamiana lines better suited to VF systems.},
}

*Nicotiana/genetics/metabolism/growth & development
*Gene Editing/methods
*Recombinant Proteins/biosynthesis/genetics/metabolism
CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Lactones/metabolism
Dioxygenases/genetics/metabolism

@article {pmid42563555,
year = {2026},
author = {Zhou, J and Wang, X and Zhang, Y and Luo, X and Quan, S and Ji, J and Sun, L and Chen, X and Chen, J and Cheng, J and Fan, J and Li, C and Cheng, B and Li, X},
title = {Natural and CRISPR/Cas9 Editing Variations of Dual-uORFs Synergistically Increase the Nutritional Value of Silage Maize Feed.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {5702-5718},
pmid = {42563555},
issn = {1467-7652},
support = {2023n06020023//Anhui Provincial Key Research and Development Program/ ; 2025SWYZ0200//Bio-breeding Laboratory of Anhui Province/ ; },
mesh = {*Zea mays/genetics/metabolism ; *Silage ; *Nutritive Value/genetics ; *CRISPR-Cas Systems/genetics ; *Open Reading Frames/genetics ; *Gene Editing/methods ; Animal Feed ; Ascorbic Acid/metabolism ; },
abstract = {The precise enhancement of nutritional quality in silage maize is a core strategy for increasing livestock production efficiency. Through evolutionary analysis of multiple plant species, we identified two functionally synergistic upstream open reading frames (uORF1 and uORF2) within the 5' untranslated region of the gene encoding GDP-L-galactose phosphorylase (GGP). By leveraging a natural translation-enhancing haplotype of uORF1 (Hap2) and performing CRISPR/Cas9-mediated targeted mutagenesis of the highly conserved uORF2, we successfully engineered an elite dual-uORFs variant. This variant significantly increased vitamin C (Vc) content and concurrently improved key silage quality traits, including crude protein and phosphorus levels, without inducing growth penalties. Transcriptomic profiling further elucidated the molecular mechanisms by which the dual-uORFs variation coordinately regulates Vc biosynthesis and the improvement of silage quality. Our findings deepen the understanding of the conventional paradigm of single-uORF regulation and provide a novel strategy and superior germplasm resources for the precision breeding of high-Vc, high-quality silage maize.},
}

*Zea mays/genetics/metabolism
*Silage
*Nutritive Value/genetics
*CRISPR-Cas Systems/genetics
*Open Reading Frames/genetics
*Gene Editing/methods
Animal Feed
Ascorbic Acid/metabolism

@article {pmid42683811,
year = {2026},
author = {Rehman, F and Liu, H and Ma, Y and Zeng, S and Wu, Y and Zong, Y and Yang, C and Wang, Y},
title = {CRISPR/Cas9-Mediated Knockout of LbSP1 and LbSP5G1 Reveals Efficient Customization of Shoot Architecture in Black Goji Berry.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {5868-5890},
pmid = {42683811},
issn = {1467-7652},
support = {32170389//National Natural Science Foundation of China/ ; 32570435//National Natural Science Foundation of China/ ; 32370399//National Natural Science Foundation of China/ ; 2023B1212060046//Guangdong Science and Technology Plan Project/ ; XDA0440000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 2022B1111230001//the Key Technologies R & D Program of Guangdong Province/ ; },
mesh = {*Plant Shoots/genetics/anatomy & histology/growth & development ; *CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; *Fruit/genetics/growth & development ; *Lycium/genetics/growth & development/anatomy & histology ; Flowers/genetics/growth & development ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Phosphatidylethanolamine Binding Protein/genetics/metabolism ; },
abstract = {Goji is recognized as a nutritionally rich superfruit with medicinal and industrial importance. However, its inherently indeterminate growth habit causes asynchronous flowering and uneven fruit ripening, resulting in harvesting inefficiency with high labour costs and low fruit yield. To elucidate the molecular basis of shoot determinacy and early flowering, comparative transcriptome analysis of indeterminate and determinate shoot apices in an F1 hybrid population of Lycium barbarum identified 15 candidate genes associated with shoot growth termination and early flowering, including an SP-like gene (LbSP1, Lba0102749) and a goji homologue of SlSP5G (LbSP5G1, Lba0501086) from the phosphatidylethanolamine-binding protein (PEBP) family. CRISPR/Cas9-mediated knockout of LbSP1 and LbSP5G1 in black goji berry produced homozygous single (CRLbSP1, CRLbSP5G1) and double determinate (2gCR LbSP1 + LbSP5G1) lines exhibiting compact shoot architecture, reduced day-length sensitivity, terminal flowering, and improved fruit yield. The double determinate line showed a significantly reduced number of leaves to the first inflorescence (7.25) compared to wild-type (WT) (18.6), with fruit yield per plant of 174.94 g, followed by CRLbSP5G1 (64.55 g), CRLbSP1 (55.17 g), and WT (22.09 g). Transcriptomic and co-expression analysis revealed extensive reprogramming of flowering regulatory networks, with LbSP1 and LbSP5G1 functioning as central regulatory hubs. Protein-protein interaction assays further established functional divergence between the two genes within a PEBP-centred network governing meristem fate. These findings provide the first functional characterization of terminal flowering genes in goji, establishing a molecular framework for breeding compact, early-flowering, and high-yield cultivars optimized for mechanized harvesting.},
}

*Plant Shoots/genetics/anatomy & histology/growth & development
*CRISPR-Cas Systems/genetics
*Plant Proteins/genetics/metabolism
*Fruit/genetics/growth & development
*Lycium/genetics/growth & development/anatomy & histology
Flowers/genetics/growth & development
Gene Expression Regulation, Plant
Gene Knockout Techniques
*Phosphatidylethanolamine Binding Protein/genetics/metabolism

@article {pmid42740447,
year = {2026},
author = {Kim, WN and Choi, YR and Kim, HJ and Chung, YS and Kim, HU},
title = {Multiplex FAD2/FATB Editing Generates Ultra-High-Oleic, Low-Saturate Soybean With Increased Seed Fatty Acid Content.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {5930-5945},
pmid = {42740447},
issn = {1467-7652},
support = {RS-2024-00410854//National Research Foundation of Korea/ ; RS-2025-00514459//National Research Foundation of Korea/ ; RS-2025-16069020//National Research Foundation of Korea/ ; RS-2024-00322277//Rural Development Administration/ ; RS-2026-25536374//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Seeds/metabolism/genetics ; *Fatty Acid Desaturases/genetics/metabolism ; *Fatty Acids/metabolism ; *Oleic Acid/metabolism ; *Gene Editing/methods ; *Thiolester Hydrolases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Oleic acid (18:1), saturated fatty acid (SFA), and polyunsaturated fatty acid (PUFA) levels are important traits for storage stability and edibility. In this study, we aimed to develop high-oleic soybean (Glycine max) by simultaneously targeting fatty acid desaturase 2 (FAD2) and fatty acyl-ACP thioesterase B (FATB) gene families using CRISPR/Cas9. Considering the paleopolyploid genome of soybean, multiple sgRNAs were designed to target GmFAD2-1, GmFAD2-2, and GmFATB genes expressed during seed development. Compared with targeting GmFAD2-1 alone (~83%), additional editing of GmFAD2-2B, GmFAD2-2C, and GmFATB1a increased the 18:1 fatty acid content to over 90%. Editing of GmFATB significantly reduced the SFA content by more than 40% compared with wild-type (WT). Reduced absolute SFA content was also observed in GmFAD2-only edited lines with decreased GmFATB expression. Notably, all evaluated high-oleic genotypes in the Williams82-background showed higher mean total fatty acid (TFA) content than the WT, both per unit seed mass and per seed. Correlation analysis of 805 individual seed profiles from Williams82-background lines further characterized the relationship between 18:1 fatty acid composition and TFA content per unit seed mass. Gene expression analysis revealed no consistent increase in triacylglycerol (TAG) synthesis genes, whereas the expression of sugar-dependent 1 (SDP1) was reduced. In contrast, genes involved in phosphatidic acid (PA) metabolism, including diacylglycerol kinase (DGK) and phospholipase D (PLD), were partially upregulated. These transcriptional changes support a hypothesis that altered lipid turnover may contribute to the higher TFA phenotype. Collectively, this study defines effective multiplex target combinations for ultra-high oleic, low-saturated soybean.},
}

*Glycine max/genetics/metabolism
*Seeds/metabolism/genetics
*Fatty Acid Desaturases/genetics/metabolism
*Fatty Acids/metabolism
*Oleic Acid/metabolism
*Gene Editing/methods
*Thiolester Hydrolases/genetics/metabolism
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
CRISPR-Cas Systems
Gene Expression Regulation, Plant

@article {pmid42804947,
year = {2027},
author = {Yang, T and Yu, X and Wu, J},
title = {Matrix conditioning and magnetic bead target transfer for fluorescence aptamer-CRISPR/Cas12a detection of kanamycin in river water.},
journal = {Talanta},
volume = {313},
number = {Pt C},
pages = {130670},
doi = {10.1016/j.talanta.2026.130670},
pmid = {42804947},
issn = {1873-3573},
mesh = {*Kanamycin/analysis ; *Rivers/chemistry ; *Aptamers, Nucleotide/chemistry ; *Water Pollutants, Chemical/analysis ; *CRISPR-Cas Systems ; Limit of Detection ; Biosensing Techniques/methods ; Fluorescence ; },
abstract = {Fluorescence aptamer-CRISPR/Cas12a assays provide efficient signal conversion and amplification for the detection of small molecules. However, their practical application in complex environmental water remains limited. Matrix components can cause signal loss before CRISPR amplification. In this study, a fluorescence aptamer-CRISPR/Cas12a assay was developed by integrating matrix conditioning and magnetic bead target transfer for kanamycin (KAN) detection in complex water matrices. Matrix conditioning reduced interference from humic acid (HA) and inorganic ions, while magnetic bead target transfer captured KAN and transferred it into a small volume of eluate. The transferred KAN induced aptamer release from aptamer/complementary strand duplexes on streptavidin magnetic beads. The released aptamer activated reporter cleavage by Cas12a and generated an increased fluorescence response. In a model matrix containing HA and inorganic ions, the assay produced only weak responses to KAN without matrix conditioning. In contrast, the workflow with matrix conditioning produced a clear increase in response with KAN concentration. In the conditioned model matrix, the assay achieved a linear response over 0.4-4 nM with a limit of detection (LOD) of 0.4 nM. This LOD was below the proposed predicted no-effect concentration (PNEC) for KAN in river water. Recoveries of 90-105% were obtained in spiked real river water samples, supporting its applicability to real water analysis. These results demonstrate that combining matrix conditioning with magnetic bead target transfer improves the matrix compatibility of fluorescence aptamer-CRISPR/Cas12a detection for trace KAN analysis in environmental water.},
}

*Kanamycin/analysis
*Rivers/chemistry
*Aptamers, Nucleotide/chemistry
*Water Pollutants, Chemical/analysis
*CRISPR-Cas Systems
Limit of Detection
Biosensing Techniques/methods
Fluorescence

@article {pmid42804951,
year = {2027},
author = {Wang, X and Dong, L and Zhang, S and Qiu, F and Wang, L and Wang, C and Wang, C and Zhu, X},
title = {One-step MCDA-CRISPR/Cas12b platform for point-of-care testing of Shewanella algae.},
journal = {Talanta},
volume = {313},
number = {Pt C},
pages = {130672},
doi = {10.1016/j.talanta.2026.130672},
pmid = {42804951},
issn = {1873-3573},
mesh = {*Shewanella/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Limit of Detection ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Shewanella algae (S. algae) is an emerging marine zoonotic pathogen capable of causing various infections with a high mortality rate. Existing detection methods for S. algae tend to be nonspecific, complicated, and time-consuming. Here, we developed a CRISPR-based detection method, named MCTOS (Multiple Cross Displacement Amplification CRISPR/Cas12b-based Testing in One-Step), for S. algae detection. Through pan-genome analysis of 193 S. algae genomes against 103 other Shewanella genomes, we identified a S. algae-specific gene designated SA, as the detection target. MCTOS combines MCDA for target pre-amplification and CRISPR/Cas12b-based cleavage for signal readout in one-pot at a constant temperature of 61 °C for 45 min. Including a 15 min rapid nucleic acid extraction step, the total assay time is 60 min with real-time fluorescence for results visualization, or 65 min with an additional 5 min lateral flow biosensor readout. The limit of detection was 10 copies, and no cross-reactivity was observed against 14 common non-S. algae templates. In evaluations of 50 isolates and 47 samples, MCTOS showed 100% (27/27) specificity and sensitivities of 98.6% (69/70) and 95.7% (67/70) for the real-time fluorescence instrument and lateral flow biosensor, respectively. Compared with recent one-pot CRISPR methods and the loop-mediated isothermal amplification method for S. algae detection, MCTOS simplifies primer design and achieves higher sensitivity. In summary, MCTOS is a simple, rapid, convenient, highly sensitive, and specific method for pathogen detection that can serve as a point-of-care testing tool for clinical diagnosis and epidemiological surveillance, and is especially suited for basic, field, and clinical laboratories.},
}

*Shewanella/genetics/isolation & purification
*CRISPR-Cas Systems
*Point-of-Care Testing
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Limit of Detection
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42810556,
year = {2026},
author = {Zainul, R and Abdul Khaleq, MA and Farah, H and Panigrahi, R and Muniyandy, E and Kumar, N and Mehla, S and Jead, OZ and Dilafruz, K and Abdullaev, A},
title = {CRISPRi-mediated silencing of efflux pump genes restores antibiotic susceptibility in multidrug-resistant Acinetobacter baumannii.},
journal = {Microbial pathogenesis},
volume = {221},
number = {},
pages = {108854},
doi = {10.1016/j.micpath.2026.108854},
pmid = {42810556},
issn = {1096-1208},
mesh = {*Acinetobacter baumannii/genetics/drug effects ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Multiple, Bacterial/genetics ; *Gene Silencing ; *Membrane Transport Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Microbial Sensitivity Tests ; *CRISPR-Cas Systems ; Gene Expression Regulation, Bacterial ; Humans ; },
abstract = {Multidrug efflux pumps are a critical mechanism underlying both intrinsic and acquired resistance in Acinetobacter baumannii. One of the most perilous multidrug-resistant (MDR) bacterial infections. Resistance-nodulation-division transporters, including AdeABC, AdeIJK, and AdeFGH, are critical efflux systems that diminish intracellular antibiotic levels, enhance bacterial persistence, support biofilm-related tolerance, and lead to treatment failure. Recent advancements in CRISPR interference (CRISPRi), a programmable gene-silencing method using catalytically inactive Cas9 (dCas9), have enabled a versatile platform for reversible, sequence-specific transcriptional repression without inducing double-strand DNA breaks. CRISPRi has emerged as an essential instrument for functional genomics, antimicrobial target validation, and the investigation of complex resistance networks in multidrug-resistant bacteria because to its precision, adaptability, and multiplexing capacities. This work primarily focuses on the recent advancements in CRISPRi-mediated silencing of efflux pump genes and their regulatory circuits in A. baumannii. Targeted suppression of key RND transporters, including adeB, adeJ, and adeG, along with regulatory components such as adeRS, has demonstrated in experiments a substantial decrease in efflux activity, an increase in intracellular antibiotic accumulation, a reduction in minimum inhibitory concentrations, and a reinstatement of susceptibility to various antimicrobial classes, including carbapenems, fluoroquinolones, aminoglycosides, tetracyclines, tigecycline, and chloramphenicol. CRISPRi-based functional screening has identified weaknesses in lipooligosaccharide synthesis, outer membrane biogenesis, and global regulatory pathways, which together augment antibiotic effectiveness with direct efflux pump inhibition. Recent research suggests that the multifactorial resistance mechanisms of MDR A. baumannii may be addressed by multiplex CRISPRi methods and combination therapies that include gene silencing with conventional antibiotics or efflux pump inhibitors. The evaluation also critically examines current delivery techniques, which will eventually determine the feasibility of translating CRISPRi medicines. Examples of contemporary platforms include plasmid-based systems, chromosomally integrated expression cassettes, conjugation-mediated transfer, bacteriophage-mediated delivery, and innovative nanotechnology-assisted carriers designed to improve intracellular delivery and bacterial targeting. Despite substantial advancements in experimental CRISPRi methodologies, the efficient delivery to infected tissues and clinical isolates remains a considerable challenge. Challenges include restricted infiltration into established biofilms, variable gene repression among genetically diverse strains, potential off-target effects, emergence of escape mutations, preservation of genetic stability, immune-mediated elimination of delivery vehicles, large-scale production and regulatory hurdles, and the absence of dependable animal and clinical studies validating therapeutic efficacy and safety. Current knowledge indicates that CRISPRi-mediated efflux pump silencing is a very promising precision antimicrobial adjuvant method that might enhance the efficacy of existing antibiotics against MDR A. baumannii. Before this technique can be integrated into regular clinical practice, substantial technological advancements, improved delivery systems, comprehensive biosafety evaluations, and rigorous in vivo and clinical validations will be required.},
}

*Acinetobacter baumannii/genetics/drug effects
*Anti-Bacterial Agents/pharmacology
*Drug Resistance, Multiple, Bacterial/genetics
*Gene Silencing
*Membrane Transport Proteins/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Microbial Sensitivity Tests
*CRISPR-Cas Systems
Gene Expression Regulation, Bacterial
Humans

@article {pmid42817230,
year = {2026},
author = {Zheng, M and Liu, R and Huang, WC and Li, Z and Hua, K},
title = {A Lambda Red Recombinase-Assisted CRISPR/Cas9 System for Efficient and Precise Genome Editing in Agrobacterium tumefaciens.},
journal = {Plant biotechnology journal},
volume = {24},
number = {11},
pages = {6010-6027},
pmid = {42817230},
issn = {1467-7652},
support = {G242402//Shanghai Landscaping and City Appearance Administration Bureau/ ; G262402//Shanghai Landscaping and City Appearance Administration Bureau/ ; YLS-2025-ZY04010//Yuelushan Laboratory Breeding Program/ ; },
mesh = {*Agrobacterium tumefaciens/genetics ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism/genetics ; *Gene Editing/methods ; Bacteriophage lambda/enzymology ; },
abstract = {Agrobacterium tumefaciens is a powerful delivery tool for plant genetic transformation and a pivotal platform for agricultural biotechnology. However, laboratory strains of Agrobacterium have remained genetically unoptimized for decades, with genome engineering hindered by inefficient, endogenous homologous recombination systems. Here, we developed and optimized a lambda (λ) Red recombinase-assisted CRISPR/Cas9 system that enables efficient and precise introduction of point mutations, insertions, deletions and large fragment replacements into both the chromosome and Ti plasmids of different A. tumefaciens strains, with minimal off-target editing. Editing efficiencies across eighteen target sites ranged from 26.7% to 100%, demonstrating the versatility of this method. Furthermore, we incorporated a SacB counter-selection marker into our system that facilitates the plasmids curing after editing. Using this platform, we sequentially deleted four antibiotic-resistance loci in strain EHA105, generating an antibiotic-sensitive derivative, EHA105-S, that maintains wild-type growth kinetics and transformation efficiency. Additionally, we engineered the microbe-associated molecular pattern (MAMPs) of two elongation factor Tu (EF-Tu) genes, allowing the A. tumefaciens to escape EF-Tu receptor (EFR) perception. The resulting engineered strains displayed enhanced transient expression efficiency in Arabidopsis thaliana. Our λ Red-assisted CRISPR/Cas9 system thus provides a robust, scalable and user-friendly tool for Agrobacterium genome engineering, paving the way for the design of next-generation strains to revolutionize plant transformation and advance synthetic biology applications in plants.},
}

*Agrobacterium tumefaciens/genetics
*CRISPR-Cas Systems/genetics
*Recombinases/metabolism/genetics
*Gene Editing/methods
Bacteriophage lambda/enzymology

@article {pmid42822295,
year = {2027},
author = {Gong, Y and Huang, X and Nong, H and Ren, H and Huang, Y and Tang, Q and Zhang, K and Liao, X},
title = {A G4-nanotweezer/DNA tetrahedron-confined dual-mode ECL/SERS biosensor for ultrasensitive rabies viral RNA detection via CRISPR-Cas12a-regulated hemin switching.},
journal = {Talanta},
volume = {313},
number = {Pt C},
pages = {130674},
doi = {10.1016/j.talanta.2026.130674},
pmid = {42822295},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *RNA, Viral/analysis/genetics ; *Hemin/chemistry ; *CRISPR-Cas Systems ; *Rabies virus/genetics/isolation & purification ; Spectrum Analysis, Raman/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Electrochemical Techniques/methods ; Luminescent Measurements/methods ; Limit of Detection ; },
abstract = {Reliable detection of low-abundance rabies viral RNA (RABV RNA) requires sensitive readouts that tolerate interfacial and matrix-associated signal variation. We developed a DNA tetrahedron-confined G4-nanotweezer biosensor combining electrochemiluminescence (ECL) and surface-enhanced Raman scattering (SERS). A Ti3C2/CsPbBr3@PDA@AuNPs nanocomposite (PAM) provided the dual-mode transducing interface, with hemin serving as both an ECL-modulating species and a SERS reporter. CsPbBr3 served as the ECL emitter, PDA improved interfacial stability, AuNPs provided SERS hotspots and DNA anchoring sites, and Ti3C2 acted as a conductive scaffold. Without target RNA, the bridging strand (ST) promoted a closed G4/hemin configuration, producing high SERS and low ECL signals. RABV RNA-triggered binding-induced isothermal amplification generated single-stranded DNA activators for CRISPR-Cas12a. Subsequent ST cleavage prevented nanotweezer closure and reduced interfacial hemin enrichment, producing an ECL-on/SERS-off response. Control experiments linked hemin-associated ECL modulation to the interfacial configuration without establishing a unique microscopic quenching pathway. The ratiometric signal R = IECL/ISERS enabled quantification over 1 aM to 50 fM. Additional low-concentration calibration yielded an estimated detection limit of 0.0675 aM by blank-threshold inverse prediction, extrapolated below the lowest measured nonzero standard of 0.10 aM. At 10 fM, matched-channel comparisons gave ratiometric relative standard deviations of 2.49% for ten successive readings and 2.87% across six independently prepared electrodes. In tenfold-diluted human serum at 10 fM, the ratio showed an 8.20% absolute deviation from phosphate-buffered saline, indicating partial matrix-effect compensation. This shared-reporter architecture links programmable DNA switching to complementary optical readouts for sensitive ratiometric viral RNA analysis.},
}

*Biosensing Techniques/methods
*RNA, Viral/analysis/genetics
*Hemin/chemistry
*CRISPR-Cas Systems
*Rabies virus/genetics/isolation & purification
Spectrum Analysis, Raman/methods
Metal Nanoparticles/chemistry
Gold/chemistry
Electrochemical Techniques/methods
Luminescent Measurements/methods
Limit of Detection

@article {pmid42933160,
year = {2027},
author = {Yu, L and Li, X and Huang, L},
title = {Cascade DNA walker-driven photo-controlled CRISPR/Cas12a nanorobot enabling ultrasensitive one-pot detection of dual miRNAs in triple-negative breast cancer.},
journal = {Talanta},
volume = {313},
number = {Pt C},
pages = {130831},
doi = {10.1016/j.talanta.2026.130831},
pmid = {42933160},
issn = {1873-3573},
mesh = {Humans ; *Triple Negative Breast Neoplasms/diagnosis/genetics/blood ; *MicroRNAs/blood/genetics/analysis ; *CRISPR-Cas Systems ; Female ; *Biosensing Techniques/methods ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Cell Line, Tumor ; Biomarkers, Tumor/blood/genetics ; },
abstract = {MicroRNAs (miRNAs) are critical biomarkers for the early diagnosis of triple-negative breast cancer (TNBC), but sensitive simultaneous detection of multiple miRNAs remains a clinical challenge. Herein, we report an innovative biosensing method based on a cascade DNA walker-driven photo-controlled CRISPR/Cas12a nanorobot for one-pot, rapid, and ultrasensitive dual miRNA detection. Target miRNAs trigger the cascade DNA walker, which integrates catalytic hairpin assembly and enzyme-mediated strand displacement, to generate abundant single-stranded DNA (ssDNA). These ssDNA then activate the photo-controlled CRISPR/Cas12a nanorobot under light illumination, enabling amplified fluorescence signaling for dual-target quantification. The assay exhibits a linear range of 100 aM to 1 pM with a limit of detection of 48.7 aM. Serum recovery experiments and cell line tests confirm its reliability in complex matrices. Clinical validation using 30 plasma samples (15 TNBC patients, 15 healthy controls) demonstrates high diagnostic accuracy (area under the curve, AUC = 0.964). This method features simple operation and strong clinical translational potential, offering a reliable tool for the early diagnosis of TNBC.},
}

Humans
*Triple Negative Breast Neoplasms/diagnosis/genetics/blood
*MicroRNAs/blood/genetics/analysis
*CRISPR-Cas Systems
Female
*Biosensing Techniques/methods
DNA, Single-Stranded/chemistry
Limit of Detection
Cell Line, Tumor
Biomarkers, Tumor/blood/genetics

@article {pmid42947239,
year = {2026},
author = {Rovina, D and Milasi, S and Di Giuseppe, G and Pioner, JM and Langione, M and Giammarino, L and Ferrantini, C and Chiesa, M and Canonico, F and D'Amario, D and Mercuri, EM and Pompilio, G and Sommariva, E and Gowran, A and Maione, AS},
title = {Phenotype rescue through the restoration of full-length dystrophin using CRISPR/Cas9 genome editing in Duchenne muscular dystrophy patient-derived iPSCs carrying the deletion of two exons.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42947239},
issn = {1756-994X},
support = {GR-2021-12375403//Ministero della Salute/ ; GR-2021-12375403//Ministero della Salute/ ; GR-2021-12375403//Ministero della Salute/ ; 2764167//Ministero della Salute/ ; 2764167//Ministero della Salute/ ; 20223L2C9N//Ministero dell'Università e della Ricerca/ ; GUP19012//Fondazione Telethon/ ; GUP19012//Fondazione Telethon/ ; JTC2018-046//European Commission ERA-NET on Cardiovascular Diseases/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Dystrophin/genetics ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *CRISPR-Cas Systems ; *Exons ; *Gene Editing ; Phenotype ; Myocytes, Cardiac/metabolism ; Fibroblasts/metabolism ; *Sequence Deletion ; Cell Differentiation ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) is a X-linked disease affecting skeletal and cardiac muscle and is caused by mutations in the dystrophin gene (DMD). Patient-derived induced pluripotent stem cells (iPSCs) serve as reliable in vitro disease models. Their genetic correction by CRISPR/Cas9 allows the generation of isogenic controls and holds promises for gene therapy. However, restoring full-length dystrophin, especially when deletions involve multiple exons, constitutes a technological challenge. This study aimed to fully repair the dystrophin gene from a DMD iPSC line carrying the deletion of exons 49-50 and to characterize the rescue of the cardiac phenotype.

METHODS: We developed an innovative CRISPR/Cas9-based approach involving the insertion of coding sequences of the deleted region, at the 3' of exon 48, thereby generating a single continuous coding sequence encompassing exons 48-49-50. Subsequently, iPSCs were differentiated into cardiomyocytes and cardiac fibroblasts. Cardiac phenotypes were analysed by western blot, immunofluorescence, ELISA, FACS, Ionoptix, 3D engineered heart tissue (EHT) and single-nuclei RNA-seq.

RESULTS: The correction of a two-exons DMD gene deletion in Duchenne iPSCs, using CRISPR/Cas9, enabled the re-expression of a stable and functional full-length dystrophin in cardiomyocytes resulting in the rescue of cardiac pathological phenotypes. Edited cardiomyocytes showed improved morphology, reduced release of the cardiomyocytes damage marker troponin I, and decreased ROS production. Moreover, dystrophin restoration enhanced contractility and ameliorated the Ca[2+] kinetics. Notably, edited iPSC derived fibroblasts showed reduced pro-fibrotic stimuli response. In parallel, we also observed enhanced functioning of a 3D engineered heart tissue and profound change in the transcriptomic profile in both cardiomyocytes and fibroblasts after the re-expression of full-length dystrophin.

CONCLUSIONS: We developed an innovative approach that enabled the re-expression of full-length dystrophin in a DMD iPSC line with consequent complete rescue of in vitro DMD cardiac phenotypes. On the long term, these results could lay a foundation for future applications of cell therapy or in vivo CRISPR/Cas9-based intervention to treat DMD.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Dystrophin/genetics
*Muscular Dystrophy, Duchenne/genetics/therapy/pathology
*CRISPR-Cas Systems
*Exons
*Gene Editing
Phenotype
Myocytes, Cardiac/metabolism
Fibroblasts/metabolism
*Sequence Deletion
Cell Differentiation

@article {pmid42947566,
year = {2026},
author = {de Souza, HCA and Dos Santos, AMP and Almeida, ACO and Conte Junior, CA and Panzenhagen, P},
title = {Type IV-Dominated Plasmid-Encoded CRISPR-Cas Systems Are Enriched in Multireplicon and Multi-Stressor Resistance Plasmids Across the Global Plasmidome.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.70132},
pmid = {42947566},
issn = {1365-2958},
support = {E26/202.227/2018//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E26/204.078/2022//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; E26/202.715/2026//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; 313119/2020-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; FinanceCode001//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
abstract = {Plasmids are central vectors of antimicrobial resistance (AMR). However, the features that distinguish the most epidemiologically successful resistance plasmids remain incompletely understood. CRISPR-Cas systems are classically regarded as barriers to horizontal gene transfer, which makes their occurrence within resistance plasmids yet unexplored at large scale. Here we analyzed 72,555 curated plasmid sequences from the Plasmid database (PLSDB) to test whether plasmid-encoded CRISPR-Cas systems are non-randomly associated with plasmid architecture and multi-stressor resistance across a large scale plasmidome. Using a three-tier classification of CRISPR-Cas system integrity based on array quality and Cas gene presence, we found that intact systems, defined by the co-occurrence of a high-confidence array and cas genes within the same replicon, were rare (1.03%) but strongly and specifically enriched in multireplicon plasmids (OR = 12.36) and in plasmids resistant to multiple stressor classes (MSR; OR = 6.25). These associations were robust to clonal redundancy: after dereplicating near-identical sequences over 100 iterations, both remained significant in every iteration, whereas the association with antimicrobial multidrug resistance (MDR) collapsed, identifying it as an artifact of clonal expansion. Intact systems were concentrated in high-risk IncF and IncH backbones and were more prevalent in plasmids from environmental sources than in those from clinical sources. Type IV-A was the predominant subtype (38.8% of intact systems), consistent with its near-exclusive association with mobile genetic elements and its proposed role in inter-plasmid competition rather than host defense. Together, these findings suggest that plasmid-encoded CRISPR-Cas systems, whether retaining defense-associated functions or acting as intra-MGE competition elements, are non-randomly retained in structurally complex, multi-stressor plasmid backbones that are particularly effective at accumulating and disseminating antimicrobial resistance.},
}

@article {pmid42947871,
year = {2026},
author = {Morfos, V and Frie, MC and Peschkov, D and Wagner, J and Lillemeier, BF and Brzostek, J},
title = {Knockout and re-expression system for mutant analysis in primary mouse T cells.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1946861},
pmid = {42947871},
issn = {1664-3224},
support = {R01 GM118879/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *T-Lymphocytes/immunology/metabolism ; Mice ; ZAP-70 Protein-Tyrosine Kinase/genetics/metabolism ; *Mutation ; CD3 Complex/genetics/metabolism ; Receptors, Antigen, T-Cell/metabolism/genetics ; Retroviridae/genetics ; CRISPR-Cas Systems ; *Gene Knockout Techniques ; Genetic Vectors/genetics ; *Gene Editing/methods ; Signal Transduction ; Cells, Cultured ; Transduction, Genetic ; },
abstract = {We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3γ and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3γ-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3γ-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.},
}

Animals
*T-Lymphocytes/immunology/metabolism
Mice
ZAP-70 Protein-Tyrosine Kinase/genetics/metabolism
*Mutation
CD3 Complex/genetics/metabolism
Receptors, Antigen, T-Cell/metabolism/genetics
Retroviridae/genetics
CRISPR-Cas Systems
*Gene Knockout Techniques
Genetic Vectors/genetics
*Gene Editing/methods
Signal Transduction
Cells, Cultured
Transduction, Genetic

@article {pmid42949637,
year = {2026},
author = {Wei, D and Ge, G and Chiang, CY and Cao, Y and Kline, ZJ and Jing, H and Golec, D and Shibata, H and Fang, D and Schwartzberg, PL and Su, HC and Zhao, K and Cheng, KC and Zhu, J},
title = {Cbfβ regulates DNA looping at the Il4-Il13 loci to control their differential expression in Th2 cells.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {12},
pages = {},
doi = {10.1084/jem.20260418},
pmid = {42949637},
issn = {1540-9538},
support = {/NH/NIH HHS/United States ; //U.S. Department of Health and Human Services/ ; NIAAI001169//Intramural Research Program/ ; ZICTR000041//Intramural Research Program/ ; ZIAHL006030//Intramural Research Program/ ; ZIAAI001059//Intramural Research Program/ ; ZIAAI001240//Intramural Research Program/ ; },
mesh = {Animals ; *Interleukin-13/genetics/metabolism ; Mice ; *Interleukin-4/genetics/metabolism ; *Core Binding Factor beta Subunit/genetics/metabolism ; Promoter Regions, Genetic ; *Th2 Cells/metabolism/immunology ; *Gene Expression Regulation ; GATA3 Transcription Factor/metabolism/genetics ; DNA/chemistry ; CRISPR-Cas Systems ; },
abstract = {Th2 cells play a central role in orchestrating type 2 immune responses by producing type 2 cytokines including IL-4 and IL-13. The master transcription factor GATA3 is essential but not sufficient for type 2 cytokine production. To better define other transcription factors that regulate type 2 cytokine expression, we performed a CRISPR-Cas9 screen after preparing a retroviral library expressing sgRNAs targeting 2,394 mouse transcription factors and cofactors. The screen revealed core-binding factor beta (Cbfβ) as a specific inducer of IL-13 that simultaneously inhibited IL-4 expression. The effect of Cbfβ on IL-13 production was confirmed both in vitro and in vivo and was independent of GATA3 induction. Mechanistically, Cbfβ induced chromatin accessibility at the HS-2 site within the Il13 promoter where the Cbfβ/Runx complex bound. Hi-TrAC analysis indicated that DNA looping between HS-2 and other enhancers was reduced upon Cbfβ deletion. Overall, our results indicate that Cbfβ directly regulates IL-13 expression by binding the Il13 promoter via the Cbfβ/Runx complex and promoting enhancer-promoter interactions that facilitate Il13 transcription.},
}

Animals
*Interleukin-13/genetics/metabolism
Mice
*Interleukin-4/genetics/metabolism
*Core Binding Factor beta Subunit/genetics/metabolism
Promoter Regions, Genetic
*Th2 Cells/metabolism/immunology
*Gene Expression Regulation
GATA3 Transcription Factor/metabolism/genetics
DNA/chemistry
CRISPR-Cas Systems

@article {pmid42950992,
year = {2026},
author = {Gupta, A and Thakur, R and Yadav, S},
title = {Nucleoredoxin proteins in green lineage: Insights about its roles in biotic and abiotic stress resilience.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {374},
number = {},
pages = {113562},
doi = {10.1016/j.plantsci.2026.113562},
pmid = {42950992},
issn = {1873-2259},
abstract = {Oxidative stress and reactive oxygen species (ROS) in plants are inevitable, and plants employ enzymatic and non-enzymatic system for cell homeostasis. Nucleoredoxins (NRX) are a family of thioredoxin (TRX) superfamily and being nuclear oxidoreductase, NRX are an important class of proteins present in many organisms including plants. Unlike TRXs, plant NRXs are larger proteins comprising of multiple TRX domains and conserved active redox motif 'WCPPC', displayed evolutionary conservation across various plants. NRX maintains oxidative stress tolerance by maintaining the reduced state by thiol-disulphide chemistry via conserved cysteine residues present in active site. Subcellular localization of NRX is mostly cytoplasmic and the putative protein interacting partners predicted their correlation with oxidative stress tolerance. We have also attempted to correlate plant NRXs with the available patent documents and observed that very little information is available. Compared to TRX research that started in 1960s, NRX have been discovered later and their structural complexity, multiple isoforms have led to their functional characterization more challenging. NRXs have roles in abiotic and biotic stresses and emerging roles indicate its role in plant immunity and modulation of plant defense response at molecular level by -OMICS studies. In view of the above, the present review discusses the sequence analysis, structure, and functional genomics associated with NRXs in plants. This is also correlated with the futuristic aspect of NRX roles being delineated in molecular stress response and signaling using CRISPR-Cas genome editing and related techniques.},
}

@article {pmid42843103,
year = {2027},
author = {Huang, D and Shi, S and Wang, X and Li, W and Zhang, X and Xiang, W and Xu, Z and He, Y and Shen, P and Fang, X and Xu, Z},
title = {A target amplification-free CRISPR biosensor with digital microfluidic processing for microRNA profiling in AI-enhanced pediatric sepsis diagnosis.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119298},
doi = {10.1016/j.bios.2026.119298},
pmid = {42843103},
issn = {1873-4235},
mesh = {Humans ; *Sepsis/diagnosis/genetics/blood ; *MicroRNAs/genetics/isolation & purification ; *Biosensing Techniques/instrumentation ; Machine Learning ; Child ; CRISPR-Cas Systems ; Microfluidic Analytical Techniques ; Equipment Design ; },
abstract = {MicroRNA signatures provide an informative readout of host-response dysregulation in pediatric sepsis, but their clinical translation remains limited by low target abundance and multiplexing requirements. Here, a target amplification-free CRISPR biosensing platform was established. The sensing mechanism couples Cas13a target recognition to promoter release, template reconstruction, and transcription-driven Cas13a reactivation, enabling fluorescence amplification through internal molecular cycling without amplifying the target miRNAs. Then, an electrowetting-based digital microfluidic chip was designed to compartmentalize parallel reactions and automate droplet generation, reaction assembly, isothermal incubation, magnetic separation, and fluorescence readout. Under optimized conditions, four sepsis-associated biomarkers (miR-15a, miR-21, miR-146a, and miR-155) and the reference control (snRNA U44) were detected at attomolar levels (expected mean input of 1.67 molecules per reaction) with good specificity and minimal inter-droplet interference. The resulting miRNA profiles were then used as input features for machine learning-enhanced sepsis classification and risk assessment. In 252 clinical samples, the resulting model achieved a test-set AUC of 0.973 and an overall accuracy of 92.1%. The model-derived risk score was associated with the Phoenix Sepsis Score and, in a representative longitudinal case, showed preliminary potential to reflect disease evolution. Overall, this platform enables automated analysis of disease-associated miRNA signatures and demonstrates its potential for in-vitro diagnosis of pediatric sepsis.},
}

Humans
*Sepsis/diagnosis/genetics/blood
*MicroRNAs/genetics/isolation & purification
*Biosensing Techniques/instrumentation
Machine Learning
Child
CRISPR-Cas Systems
Microfluidic Analytical Techniques
Equipment Design

@article {pmid42843104,
year = {2027},
author = {Li, X and Guo, Z and Ye, W and Chai, H and Miao, P},
title = {Ligation-assisted DNA strand displacements and CRISPR/Cas14 cleavage for ultrasensitive detection of PIWI-interacting RNA.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119308},
doi = {10.1016/j.bios.2026.119308},
pmid = {42843104},
issn = {1873-4235},
mesh = {*Piwi-Interacting RNA/genetics/analysis/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/diagnosis ; DNA/chemistry ; *RNA, Small Interfering/genetics ; Nucleic Acid Amplification Techniques/methods ; Female ; },
abstract = {PIWI-interacting RNA (piRNA) as a type of small non-coding RNA holds great promise for managing disease occurrence and progression. Herein, we construct a ligation-assisted DNA strand displacement strategy for enhanced CRISPR/Cas14 cleavage, which provides remarkable SERS and electrochemical responses to indicate trace piRNA information. The upstream reaction is rationally designed to integrate traditional strand displacement amplification (SDA) and ligation-mediated target recycling, which benefits improvement of the efficiency. The generated single-stranded DNA products can activate trans-cleavage activity of Cas14, enabling the DNA structural changes on the sensing interface and subsequent signal measurements. By coupling ligation-assisted SDA (LSDA) and CRISPR/Cas14 system, ultrahigh sensitivity is achieved with low background signal. This method not only reports consistent results comparing with standard qRT-PCR technique, but can discriminates piRNA expressions in samples from breast cancer patients. Therefore, a reliable and practical tool is provided for piRNA related clinical applications.},
}

*Piwi-Interacting RNA/genetics/analysis/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Breast Neoplasms/genetics/diagnosis
DNA/chemistry
*RNA, Small Interfering/genetics
Nucleic Acid Amplification Techniques/methods
Female

@article {pmid42858624,
year = {2027},
author = {Lee, EY and Lee, DG and Kwon, JS and Kim, SH and Shin, Y},
title = {Cleavage-gated Cas13-to-DNA nanostructure transduction for target-preamplification-free viral RNA detection.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119326},
doi = {10.1016/j.bios.2026.119326},
pmid = {42858624},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *RNA, Viral/genetics/isolation & purification/analysis/chemistry ; DNA Nanostructures/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/chemistry/genetics ; },
abstract = {CRISPR-Cas13a enables programmable RNA recognition without target nucleic-acid preamplification, but conventional assays encode collateral RNase activity by cleaving fluorescent reporters. Here, we developed a cleavage-gated strategy that converts transient Cas13a cleavage into a sequence-defined, strand-displacement-competent DNA actuator, shifting signal encoding from cumulative reporter intensity to a discrete scaffold topology. A chimeric masking hairpin (CMH) links Cas13a to a DNA nanostructure sensor (DNA-NS) by using a cleavable RNA loop to constrain a DNA-key bearing a pre-exposed toehold. Target-activated Cas13a cleaves the loop, converting the partially masked key into a strand-displacement-competent intermediate that reconfigures an M13mp18 scaffold from a linear to a looped state, producing a distinct electrophoretic mobility signature. Screening of 40 CMH-sensor-handle combinations identified a 4-nt toehold paired with an 18-nt sensor handle as the best-performing configuration within the tested design space (component-level ΔΔG = -1.19 kcal/mol at 37 °C). Using pooled crRNAs targeting the S, M, and L genomic segments of severe fever with thrombocytopenia syndrome virus (SFTSV), the assay detected the lowest tested input concentration of 10.38 fM in all three replicates. In a retrospective case-control evaluation of 87 plasma-derived RNA samples, the assay agreed with RT-qPCR for 45 of 47 positive specimens and 40 healthy controls, yielding a positive percent agreement of 95.7% and a negative percent agreement of 100%. Replacing the crRNAs redirected recognition to Hantaan virus reference RNA without redesigning the CMH or DNA-NS. This work establishes a modular framework for topology-resolved viral RNA detection without target preamplification.},
}

*Biosensing Techniques/methods
*RNA, Viral/genetics/isolation & purification/analysis/chemistry
DNA Nanostructures/chemistry
*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/chemistry/genetics

@article {pmid42858625,
year = {2027},
author = {Cha, S and Zhang, P and Hou, J and Wang, L and Yang, Q and Liu, X and Yu, G and Zhang, L},
title = {Rationally optimized split crRNA-based CRISPR/Cas12a platform for amplification-free miRNA detection.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119313},
doi = {10.1016/j.bios.2026.119313},
pmid = {42858625},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification/blood/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; *Breast Neoplasms/genetics/diagnosis/blood ; Limit of Detection ; Female ; },
abstract = {MicroRNAs (miRNAs) are important biomarkers for cancer diagnosis and prognosis, but their short length, low abundance, and high sequence similarity make rapid and sensitive detection challenging. Split crRNA-based CRISPR/Cas12a systems have emerged as a promising amplification-free strategy for RNA detection, yet the design principles governing their performance remain largely unexplored. Here, we report a rationally engineered split crRNA-Cas12a platform for direct detection of breast cancer-associated miRNAs. Using miR-155 and miR-10b as model targets, the influence of spacer split position on assay performance was systematically evaluated and further interpreted through structural prediction analysis. The results revealed distinct effects of spacer split position on signal output, reaction kinetics, and background signal, providing guidance for the rational design of split crRNA-Cas12a systems. DNA activator design, Cas12a/crRNA ratio, and reaction conditions were further optimized to improve assay performance. The system enabled sensitive detection of miR-155 and miR-10b, achieving limits of detection of 68 and 98 fM, respectively, while maintaining excellent specificity against non-target miRNAs and strong discrimination of mismatched sequences. The assay supported dual-target screening within a single reaction and demonstrated reliable performance in serum-spiked samples. These findings establish key design principles for split crRNA-Cas12a systems and provide a rational framework for engineering amplification-free RNA detection platforms. The proposed platform extends the application of Cas12a for direct RNA detection and provides a promising strategy for rapid biomarker screening in point-of-care and resource-limited settings.},
}

*MicroRNAs/genetics/isolation & purification/blood/analysis
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
*Breast Neoplasms/genetics/diagnosis/blood
Limit of Detection
Female

@article {pmid42858626,
year = {2027},
author = {Tang, S and Liao, X and Chen, J and Zhou, J and Xiong, Y and Hu, B and Yang, C and Qu, J and Wang, J},
title = {Programmable thermoregulation of the nuclease activity of LbCas12a.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119310},
doi = {10.1016/j.bios.2026.119310},
pmid = {42858626},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Mycobacterium tuberculosis/isolation & purification/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; Humans ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics ; DNA, Single-Stranded/chemistry/genetics ; Temperature ; Limit of Detection ; },
abstract = {CRISPR-Cas12a holds great promise for molecular diagnostics and gene editing due to its sequence-specific DNA recognition and robust trans-cleavage activity, yet precise tuning of its nuclease activity remains challenging. Herein, we report a simple temperature-regulation strategy using cost-effective ssDNA blockers, that enable hybridize with crRNA functional motifs at low temperatures to block and suppress Cas12a activation, while thermal dissociation at elevated temperatures restores Cas12a activity to realize thermoregulation of Cas12a activity, enabling precise thermal switching within 5 °C (37-42 °C). The cis- and trans-cleavage activity of Cas12a could be activated at desired temperature via different DNA blockers. Coupling this thermoregulated Cas12a system with recombinase polymerase amplification (RPA), we developed a one-tube thermal-start RPA-CRISPR/Cas12a assay, which avoids premature Cas12a activation. This method achieves a limit of detection (LOD) of 0.73 aM against Mycobacterium tuberculosis (MTB) with excellent specificity and reproducibility, and exhibits 96.67% concordance with qPCR in clinical specimens. An all-in-one THERMAL-CRISPR POCT platform was developed by using a 3D-printed portable device for streamlined clinical testing, with clinical sensitivity of 90.48% and 100% specificity. This programmable thermal modulation strategy provides a novel and simple technical route for constructing robust, engineerable CRISPR tools for point-of-care diagnostic and gene editing.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Mycobacterium tuberculosis/isolation & purification/genetics
*CRISPR-Associated Proteins/chemistry/genetics
Humans
*Endodeoxyribonucleases/chemistry/genetics/metabolism
*Bacterial Proteins/chemistry/genetics
DNA, Single-Stranded/chemistry/genetics
Temperature
Limit of Detection

@article {pmid42869985,
year = {2026},
author = {Yu, H and Wu, Y and Liu, Y and Wei, R and Zhou, Z and Yu, L and Lin, X and Wang, J},
title = {CRISPRa screen unveils noncanonical NF-κB to boost the antitumor activity of TCR-based CAR-T cells.},
journal = {Cellular & molecular immunology},
volume = {23},
number = {11},
pages = {1513-1528},
pmid = {42869985},
issn = {2042-0226},
support = {82293664//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82341212//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31930039//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Animals ; *NF-kappa B/metabolism ; *Immunotherapy, Adoptive/methods ; Transcription Factor RelB/metabolism ; *Receptors, Antigen, T-Cell/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/metabolism ; Signal Transduction ; T-Cell Exhaustion ; *Neoplasms/immunology/therapy ; },
abstract = {Engineering autologous T cells for adoptive therapies has revolutionized cancer treatment, yet limited persistence remains a major barrier to robust clinical responses and durable disease control. Although costimulation with 4-1BB markedly improves CAR-T-cell persistence, no comparably effective approach has been identified to enhance persistence in T-cell therapies that rely on native TCR signaling, including conventional TCR-T cells and synthetic T-cell receptor and antigen receptor (STAR) T cells. To identify intrinsic persistence regulators in a TCR-based context, we performed an in vivo CRISPR activation screen in STAR-T cells and identified the noncanonical NF-κB axis, with RELB as the top hit. Direct RELB overexpression, however, exhibited dose-dependent cytotoxicity, motivating a translational alternative: overexpression of p52, the ncNF-κB transcription partner of RELB. Under prolonged antigen exposure, p52 overexpression sustained proliferation, preserved effector functions, and increased the intratumoral trafficking potential, particularly in CD8[+] T cells, thereby yielding superior tumor control. Mechanistically, transcriptomic profiling revealed coordinated upregulation of cell cycle programs and chemokine receptors, along with downregulation of apoptosis and exhaustion-associated modules. Moreover, p52 overexpression restored RELB nuclear translocation in the context of PD-1/PD-L1 engagement, indicating partial resistance to checkpoint-mediated suppression and sustained ncNF-κB activity in PD-L1[hi] immunosuppressive environments. Collectively, our findings identify p52 as an antigen-dependent enhancer of ncNF-κB, conferring durable persistence and checkpoint resilience on TCR-based T cells and providing a feasible strategy to improve therapeutic effects in solid tumors.},
}

Humans
Animals
*NF-kappa B/metabolism
*Immunotherapy, Adoptive/methods
Transcription Factor RelB/metabolism
*Receptors, Antigen, T-Cell/metabolism
*CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
*Receptors, Chimeric Antigen/metabolism
Signal Transduction
T-Cell Exhaustion
*Neoplasms/immunology/therapy

@article {pmid42874256,
year = {2027},
author = {Yang, X and Zeng, X and Qi, G and Wang, J and Wei, X and Hu, Q and Huang, J and Chen, Y and Xie, Y and Li, Y and Zhong, Y and Li, S and Yang, X},
title = {Unified hybrid RNA-regulated CRISPR diagnostic for concurrent Mycobacterium tuberculosis detection and rifampicin resistance profiling.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119329},
doi = {10.1016/j.bios.2026.119329},
pmid = {42874256},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/genetics/isolation & purification/drug effects ; *Rifampin/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins/genetics ; *Tuberculosis/microbiology/diagnosis ; Antibiotics, Antitubercular/pharmacology ; Mutation ; Limit of Detection ; DNA-Directed RNA Polymerases/genetics ; },
abstract = {CRISPR-based nucleic acid detection is challenged by protospacer adjacent motif (PAM) dependency, amplicon consumption and single-tube multiplexing barriers, and poor discrimination of single-nucleotide variations (SNVs). These limitations are particularly acute in detecting Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance mutations for tuberculosis (TB) diagnostics. Herein, we devised TB-SCANER, a broad-spectrum, one-pot CRISPR-Cas12a diagnostic platform for MTB detection and RIF resistance profiling. The assay employs two unlocking single-stranded RNAs (uLsRNAs) transcribed from RPA products to unlock four cis-cleavage-locked CRISPR-Cas12a systems via distinct locking single-stranded RNAs (LsRNAs), establishing a two-to-four hybrid logic: one uLsRNA carries a single unlocking module for the M channel (targeting mpb64 gene), whereas the other contains three tandem modules engaging LsRNAs for subregions R1, R2, R3 within rpoB gene. With genomic DNA, detection limits are 50 fg/reaction (∼10 copies) for mpb64 and 500 fg/reaction (∼100 copies) for rpoB. The assay reliably identified clinically dominant rpoB mutations (Leu511Pro, Asp516Gly, His526Tyr, Ser531Leu) and low-prevalence variants (Ser522Met, His526Arg, Leu533Pro). For cultured isolates, TB-SCANER was 100% concordant with Sanger sequencing for RIF resistance mutation profiling; in clinical samples, it matched Xpert for MTB detection (100%), outperformed culture sensitivity (100% vs. 93.8%), and was fully concordant with Sanger sequencing for RIF resistance, eliminating Xpert's false-positive while identifying all resistant strains. Collectively, TB-SCANER is a rapid, accurate, low-cost, one-pot CRISPR diagnostic that overcomes multiplexing barriers, enabling concurrent MTB detection and RIF resistance profiling particularly in resource-limited settings, and features built-in adaptability to other pathogens.},
}

*Mycobacterium tuberculosis/genetics/isolation & purification/drug effects
*Rifampin/pharmacology
*Drug Resistance, Bacterial/genetics
Humans
CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Bacterial Proteins/genetics
*Tuberculosis/microbiology/diagnosis
Antibiotics, Antitubercular/pharmacology
Mutation
Limit of Detection
DNA-Directed RNA Polymerases/genetics

@article {pmid42887285,
year = {2027},
author = {Wan, C and Liu, W and Pan, S and Wang, X and Zhou, J and Hu, Y and Li, P and Nie, X and Liao, Y},
title = {DNA assisting CRISPR-Cas13a activation regulating assay for detection of DNA mutation and miRNAs.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119344},
doi = {10.1016/j.bios.2026.119344},
pmid = {42887285},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Mutation ; *DNA/genetics/chemistry ; DNA, Single-Stranded/chemistry/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; DNA Mutational Analysis/methods ; },
abstract = {Cas13a is an RNA-targeting effector system with exceptional sensitivity, essential for applications such as mutation discrimination, RNA biomarker quantification and dynamic monitoring. However, precise control over its activation kinetics and thermodynamics remains a fundamental challenge, limiting its use in nucleic acid detection. Here, we present the DNA-Assisting CRISPR-Cas13a Activation Regulating (DACAR) Assay, which employs a single-stranded DNA blocker to occupy the crRNA spacer. Through programmable toehold-mediated strand displacement (TMSD) and toehold-exchange (TE) reactions, DACAR is able to control the thermodynamics and kinetics of Cas13a activation at two different levels. Systematic modulation of toehold length and direction yields a tunable cleavage rate gradient, while TE-based free-energy tuning facilitates an ON/OFF gating mode. For single-nucleotide variant (SNV) discrimination, DACAR-TE amplifies thermodynamic mismatch effects, achieving a discrimination factor of 162.4 and a detection limit of 0.05% mutant abundance for the KRAS G12D mutation. The Switch-DACAR system also enables sequential detection of miR-155 and miR-21 in a single tube, with a detection limit of 10 pM. To sum up, DACAR provides a unified, programmable framework for precise molecular-level control of Cas13a, advancing its potential in diagnostics and molecular logic.},
}

*MicroRNAs/genetics/analysis/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Mutation
*DNA/genetics/chemistry
DNA, Single-Stranded/chemistry/genetics
Proto-Oncogene Proteins p21(ras)/genetics
DNA Mutational Analysis/methods

@article {pmid42892233,
year = {2027},
author = {Zhao, J and Gao, M and Ding, H and Song, Y and Zhang, Y and Ten, Y and Wang, F and Miao, Z and Zhang, J and Yang, S and Jiao, J},
title = {Unipoint-anchored cascade amplification strategy integrating FUSE probe-activated CRISPR-Cas12a for dual mode osteopontin detection toward osteosarcoma assessment and therapeutic response evaluation.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119343},
doi = {10.1016/j.bios.2026.119343},
pmid = {42892233},
issn = {1873-4235},
mesh = {*Osteopontin/blood/genetics ; *Osteosarcoma/genetics/drug therapy/blood/diagnosis ; *Biosensing Techniques/methods ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Bone Neoplasms/drug therapy/genetics/blood/diagnosis ; Gold/chemistry ; Limit of Detection ; Bacterial Proteins/genetics/chemistry ; Metal Nanoparticles/chemistry ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Reliable assessment of disease status and therapeutic response in osteosarcoma remains an unmet clinical need. Integrative bioinformatics analysis and protein-level verification in paired clinical specimens prioritized secreted phosphoprotein 1 (SPP1, osteopontin) as a liquid biopsy target associated with osteosarcoma progression and patient survival. On this basis, CRUMP, a CRISPR-Cas12a and FUSE Probe-based dual-mode biosensing platform, was developed for SPP1 quantification. A bifunctional NH2-ZIF-8 probe co-immobilizes the components required for target recognition and Cas12a activation, while MoS2@AuNPs interfaces transduce the resulting signals. Signal amplification is achieved through a unipoint-anchored multisite cascade based on a DNA tetrahedron-HCR architecture, enabling one-to-multiple parallel amplification while balancing stability and sensitivity. A single Cas12a cleavage event simultaneously decreases the electrochemical (EC) signal and increases the fluorescence (FL) signal. These reciprocal changes allow the two readouts to provide an internal consistency check for each measurement. Each channel covers more than five orders of magnitude, with detection limits of 167.7 fg/mL for EC and 331.6 fg/mL for FL. The broad measurement ranges allow most serum samples to be analyzed directly, without the per-sample dilution optimization that ELISA requires, and the complete analysis takes only approximately 85 min. Validation across osteosarcoma cell lines, clinical specimens, and tumor-bearing mouse models provided preliminary evidence for the feasibility of CRUMP in SPP1 quantification and endpoint assessment of chemotherapeutic response. By combining biomarker prioritization, biosensor engineering, and preliminary application testing, this work presents CRUMP as a candidate platform for SPP1-based assessment of osteosarcoma and its therapeutic response.},
}

*Osteopontin/blood/genetics
*Osteosarcoma/genetics/drug therapy/blood/diagnosis
*Biosensing Techniques/methods
Humans
Animals
CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
*Bone Neoplasms/drug therapy/genetics/blood/diagnosis
Gold/chemistry
Limit of Detection
Bacterial Proteins/genetics/chemistry
Metal Nanoparticles/chemistry
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42913300,
year = {2027},
author = {Xiong, Y and Li, L and Huang, J and Shen, XA and Sun, J and Guo, Y and Liu, D and Ma, T and Huang, X and Xiong, Y},
title = {Target-triggered exponential amplification-enhanced CRISPR/Cas12a system with a fluorescence-quenching lateral flow assay for ultrasensitive detection of Salmonella typhimurium.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119351},
doi = {10.1016/j.bios.2026.119351},
pmid = {42913300},
issn = {1873-4235},
mesh = {*Salmonella typhimurium/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *DNA, Bacterial/genetics/analysis ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Humans ; Gold/chemistry ; Fluorescence ; Food Microbiology ; Spectrometry, Fluorescence ; },
abstract = {The use of the CRISPR/Cas12a system combined with lateral flow assays shows significant promise for point-of-care testing in resource-limited regions. However, the use of isothermal amplification technologies to preenrich target DNA fragments inherently increases the risk of aerosol contamination during strip testing. Here, we introduce a target-triggered exponential amplification-enhanced CRISPR/Cas12a system combined with a fluorescence-quenching lateral flow assay (TEA-FQLFA) for ultrasensitive detection of Salmonella typhimurium. The TEA system converts a single Cas12a recognition event into sequential dual Cas12a activation, initiating a cleavage cascade amplification that achieves a 10[6]-fold signal enhancement without the need for a target DNA preamplification operation. Moreover, hyperbranched gold-coated polymer microspheres (HGPMs), characterized by ultrahigh extinction coefficients and a broad absorption band in the 300-800 nm range, are utilized as fluorescence quenching reporters of the FQLFA for simultaneous colorimetric visualization and fluorescence-based quantitative detection of target DNA concentration. By leveraging the synergistic integration of TEA-mediated signal amplification and HGPM-enabled optical signal enhancement, the TEA-FQLFA system achieves attomolar-level detection of target DNA (0.081 aM) and enables sensitive detection of Salmonella typhimurium in complex food matrices, with detection limits of 7.95 CFU/mL in colorimetric mode and 6.94 CFU/mL in fluorescence mode. In summary, this work establishes a versatile platform that integrates cascade signal amplification with dual-mode lateral-flow for ultrasensitive food safety detection.},
}

*Salmonella typhimurium/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*DNA, Bacterial/genetics/analysis
Limit of Detection
Nucleic Acid Amplification Techniques/methods
Humans
Gold/chemistry
Fluorescence
Food Microbiology
Spectrometry, Fluorescence

@article {pmid42925592,
year = {2027},
author = {Wang, X and Shi, X and Zheng, X and Ding, Y and Jing, Y and Zhang, J and Wang, J and Li, H and Zhao, C},
title = {A DNAzyme-activated engineered crRNA enables CRISPR-Cas12a biosensing of ALP and S. aureus.},
journal = {Biosensors & bioelectronics},
volume = {316},
number = {},
pages = {119360},
doi = {10.1016/j.bios.2026.119360},
pmid = {42925592},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *DNA, Catalytic/chemistry ; *CRISPR-Cas Systems/genetics ; *Staphylococcus aureus/isolation & purification ; *Alkaline Phosphatase/isolation & purification/analysis ; Manganese/chemistry ; Limit of Detection ; Humans ; CRISPR-Associated Proteins/chemistry/genetics ; Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {CRISPR-Cas12a biosensing is hindered by insufficient sensitivity that necessitates pre-amplification, as well as the limited ability to directly detect non-nucleic-acid targets. Herein, we developed a CRISPR-Cas12a biosensor centered on a DNAzyme-activated engineered crRNA (e-crRNA), enabling Mn[2+]-responsive control over its trans-cleavage activity for nucleic-acid-amplification-free detection. Upon Mn[2+]-dependent activation, the DNAzyme cleaved the e-crRNA to liberate a full-sized crRNA1 and a split crRNA2. The combined crRNA1/crRNA2 system enhanced Cas12a trans-cleavage activity, compensating for the activity loss associated with crRNA engineering. Rational design of the split site further improved the signal-to-noise ratio. Moreover, by gating the system activation on Mn[2+], the non-specific activation effects caused by spurious DNA hybridization were mitigated. When coupled with a Mn[2+]-responsive signal conversion strategy, this platform achieved sensitive detection of alkaline phosphatase with the LOD of 0.008 U/L and Staphylococcus aureus with the LOD of 29.76 CFU/mL. This study showcased the innovative use of Mn[2+]-dependent DNAzyme to efficiently activate CRISPR-Cas12a system, establishing a powerful platform for biosensing.},
}

*Biosensing Techniques/methods
*DNA, Catalytic/chemistry
*CRISPR-Cas Systems/genetics
*Staphylococcus aureus/isolation & purification
*Alkaline Phosphatase/isolation & purification/analysis
Manganese/chemistry
Limit of Detection
Humans
CRISPR-Associated Proteins/chemistry/genetics
Staphylococcal Infections/microbiology/diagnosis

@article {pmid42944055,
year = {2026},
author = {Zhu, M and Sun, Z and Xu, K and Ye, L},
title = {Systematic Review and Meta-Analysis of Genome-Wide CRISPR-Cas Screens to Identify Host Factors Involved in HIV-1 Infection.},
journal = {Viruses},
volume = {18},
number = {10},
pages = {},
pmid = {42944055},
issn = {1999-4915},
support = {20241029Y081//the Guidance Program for the Agricultural and Social Development Research of Hangzhou/ ; 2025JK043//the Zhejiang Provincial Science and Technology Program for Disease Prevention and Control/ ; },
mesh = {Humans ; *HIV-1/physiology/genetics ; *HIV Infections/virology/genetics ; *CRISPR-Cas Systems ; *Host-Pathogen Interactions/genetics ; Virus Replication ; Virus Latency ; Receptors, CCR5/genetics ; Genome-Wide Association Study ; },
abstract = {Human immunodeficiency virus type 1 (HIV-1) replication depends on a complex interplay between viral proteins and host cellular factors, which represent promising therapeutic targets. However, systematic integration of results from genome-wide CRISPR screens for HIV host factors remains limited. Here, we performed a systematic review and meta-analysis of pooled genome-wide CRISPR-Cas9 screens to identify host dependency, restriction, and latency-promoting factors in HIV-1 infection. Following PRISMA guidelines, we identified 11 eligible studies from which we extracted 26 gene lists. These were classified into three phenotypic categories, including dependency (5 lists), restriction (10 lists), and latency-promoting (7 lists), and integrated using meta-analysis by information content (MAIC). GO over-representation analysis and Gene Set Enrichment Analysis were performed on MAIC-ranked gene lists to characterize pathway architectures. MAIC successfully recovered well-established host factors, including the entry receptors CD4, CCR5, and CXCR4; the integration cofactor PSIP1 (LEDGF/p75); and the restriction factors TRIM5, TRIM22, PML, G3BP1, and EIF2AK2. RNF114 emerged as the top-ranked restriction factor, supported by four independent publication-level categories. Additional high-confidence hits included TRIM56, BIRC3, DTX3L, and components of the ubiquitin-proteasome system across the dependency and latency datasets, further supporting the robustness of the MAIC framework. Beyond these canonical factors, we identified multiple undercharacterized genes and pathways, including coagulation and IL-17 signaling in dependency factors, SUMOylation and DNA damage response in restriction factors, and the Integrator complex and proteasome-granule co-enrichment in latency-promoting factors. Leading-edge analysis confirmed that most of these non-canonical enrichments are driven by coherent gene modules rather than a small set of pleiotropic hubs. These findings reveal distinct pathway architectures across the three infection states and highlight previously unrecognized host processes that may serve as targets for antiviral therapy. Our analysis provides a comprehensive resource for guiding future mechanistic studies of host-HIV interactions and developing strategies to enhance immunity, block viral replication, and reverse latency.},
}

Humans
*HIV-1/physiology/genetics
*HIV Infections/virology/genetics
*CRISPR-Cas Systems
*Host-Pathogen Interactions/genetics
Virus Replication
Virus Latency
Receptors, CCR5/genetics
Genome-Wide Association Study

@article {pmid42944119,
year = {2026},
author = {Hu, Z and Hailai, A and He, H and Yang, C and Chen, Y and Huang, Z and Li, X and Xu, S and Guan, R},
title = {Advances in Diagnostic Technologies of Major Avian Respiratory Viruses: A Review.},
journal = {Veterinary sciences},
volume = {13},
number = {10},
pages = {},
pmid = {42944119},
issn = {2306-7381},
support = {2026C04018//the "Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; JSYJ-2025-02//the Xichang Municipal Technology Research, Development and Promotion Application Project funded by the Xichang Municipal Bureau of Economy, Information Technology and Science/ ; 25KJFZ0011//Liangshan Science and Technology Program/ ; YBZ2025004//Doctoral Research Initiation Project of Xichang University/ ; },
abstract = {Avian respiratory diseases, particularly those induced by the avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV), represent a substantial economic threat to the global poultry industry. Several of these viruses can also transmit across species, posing risks to public health security. Given the challenges posed by similar clinical symptoms from different pathogens, the prevalence of co-infections, and rapid viral mutations, rapid, precise, and highly sensitive diagnostic techniques are imperative for effective disease prevention and control, epidemic surveillance, and epidemiological research. This review provides an overview of molecular and immunological diagnostic techniques for major avian respiratory viruses. All covered technologies are evaluated and compared across key performance metrics: target type, analytical sensitivity, multiplex capacity, turnaround time, point-of-care diagnostic techniques (POCT) suitability and application scenarios. Specifically, the review details real-time quantitative PCR (qPCR) and its multiplex formats, isothermal amplification methods including loop-mediated isothermal amplification (LAMP), recombinase-aided amplification (RAA) and recombinase polymerase amplification (RPA), and innovative CRISPR-Cas-based nucleic acid detection systems. It also examines high-throughput sequencing, microarray technologies, and advanced immunological assays enhanced by nanomaterials, recombinant proteins, and single-domain antibodies. The review further discusses integrated, automated, and portable diagnostic platforms, including microfluidics, and smartphone-assisted systems. Finally, the review outlines future trends in avian diagnostic technologies, providing a practical reference for building more efficient and accessible avian disease surveillance and early warning systems.},
}

@article {pmid42946710,
year = {2026},
author = {Liu, K and Li, X and Zhao, M and Yang, Z and Wen, J and Liu, J and Zhang, H and Zhao, Y and Sun, Y and Li, Z},
title = {A CRISPR-based 'dam-break drainage' sensing architecture for rapid, affordable and ultrasensitive pathogen detection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42946710},
issn = {2041-1723},
support = {22234001//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*RNA, Viral/genetics/analysis ; *SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; Influenza A virus/genetics/isolation & purification ; Influenza B virus/genetics/isolation & purification ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; *COVID-19 Nucleic Acid Testing/methods/economics ; },
abstract = {Nucleic acid testing is important for infectious-disease diagnosis, but combining high sensitivity with accessible testing remains challenging. Here we show a chip-based visual detection strategy for one or two viral RNA targets that involves only sample loading, without further instrumentation or manual actuation. Target-activated CRISPR-Cas13a alters the wettability of a molecular barrier, causing liquid to cross a preset threshold and generate a dam-break drainage signal. The chip detects a single target at 10 aM within 2 min and two targets at 100 aM within 5 min, with costs of $0.20 and $0.30 per test, respectively. We further establish a practical clinical-sample workflow combining room-temperature lysis with RNase inhibition and validate the platform across 40 nasal-swab samples tested for SARS-CoV-2, influenza A virus and influenza B virus, with results concordant with RT-PCR. This threshold-based fluidic strategy provides a route towards rapid, low-cost and instrument-free molecular testing outside centralized laboratories.},
}

*RNA, Viral/genetics/analysis
*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*COVID-19/diagnosis/virology
Influenza A virus/genetics/isolation & purification
Influenza B virus/genetics/isolation & purification
Rapid Diagnostic Tests
Sensitivity and Specificity
*COVID-19 Nucleic Acid Testing/methods/economics

@article {pmid42800154,
year = {2027},
author = {Yang, T and Tian, Z and Guan, Z and Wang, L and Li, B and Hu, X and Zhou, P and Chen, L and Ju, S and He, Y and Jiang, Y and Du, Y},
title = {Quantitative detection of the diabetic retinopathy-associated microRNA-1281 via a CRISPR/Cas12a-RCA ratiometric sensing platform.},
journal = {Talanta},
volume = {313},
number = {Pt B},
pages = {130655},
doi = {10.1016/j.talanta.2026.130655},
pmid = {42800154},
issn = {1873-3573},
mesh = {Humans ; *Diabetic Retinopathy/genetics/diagnosis/blood ; *MicroRNAs/genetics/blood ; *CRISPR-Cas Systems ; Benzothiazoles/chemistry ; *Bacterial Proteins/genetics ; Biomarkers/blood ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics ; Biosensing Techniques/methods ; Endodeoxyribonucleases ; },
abstract = {Diabetic retinopathy (DR) is a major microvascular complication of diabetes, yet accessible molecular tools for early-stage screening remain limited. Circulating microRNAs are promising non-invasive biomarkers for DR, but their reliable quantification in serum is hindered by low abundance, matrix interference, and limited robustness of single-signal assays. Here, we report a Cas12a-RCA ratiometric sensing platform (CRRSP) for the quantitative detection of the DR-associated biomarker miR-1281. The assay is constructed using a target-triggered cascaded strand-displacement circuit that enforces sequential molecular recognition prior to amplification, thereby improving specificity. Upon target recognition, rolling circle amplification (RCA) generates DNA products that simultaneously produce a thioflavin T (ThT) fluorescence signal via G-quadruplex (G4) formation and sequester the Cas12a/gRNA Activator strand to suppress ROX fluorescence. This dual-channel ThT/ROX readout enables intrinsic ratiometric normalization, effectively compensating for matrix effects and experimental variability. The workflow operates under sequential constant-temperature steps without PCR-type thermal cycling. The assay achieves a detection limit of 0.83 pM with high selectivity and shows excellent agreement with RT-qPCR in clinical serum samples from healthy controls, glaucoma patients, and patients with non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR).},
}

Humans
*Diabetic Retinopathy/genetics/diagnosis/blood
*MicroRNAs/genetics/blood
*CRISPR-Cas Systems
Benzothiazoles/chemistry
*Bacterial Proteins/genetics
Biomarkers/blood
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Associated Proteins/genetics
Biosensing Techniques/methods
Endodeoxyribonucleases

@article {pmid42833528,
year = {2026},
author = {Zhang, J and Yang, Y and Wu, Y and Shi, M and Tan, X and Zhang, C and Ni, Q and Wang, J and Zhang, Z and Li, Y and Zhang, K},
title = {Terminal-overhang-restricted CRISPR logic nanodevice for precision cancer therapy.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {399},
number = {Pt B},
pages = {115433},
doi = {10.1016/j.jconrel.2026.115433},
pmid = {42833528},
issn = {1873-4995},
mesh = {Animals ; Humans ; *MicroRNAs/genetics ; Mice, Nude ; *DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/metabolism ; Photochemotherapy/methods ; *Neoplasms/therapy/genetics/drug therapy ; Cell Line, Tumor ; Precision Medicine ; *CRISPR-Cas Systems ; *Antineoplastic Agents/administration & dosage ; Reactive Oxygen Species/metabolism ; Drug Liberation ; Mice ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The limited precision of controlled drug release remains a major challenge for precision cancer therapy. Herein, a protein and nucleic acid dual-biomarker-responsive CRISPR AND logic nanodevice, termed LogiCas, is developed by integrating APE1 and miRNA-21 as dual inputs for precise tumor therapy. Leveraging the stringent conformational matching required for Cas12a activation, LogiCas utilizes the terminal redundancy restriction of the split activator to establish a dual-lock, stepwise activation mechanism consisting of APE1-mediated pre-unlocking and miRNA-21-mediated terminal activation, enforcing tightly gated control over Cas12a trans-cleavage. The activated Cas12a further cleaves Ce6-BHQ3 to trigger Ce6-mediated photodynamic therapy (PDT), generating reactive oxygen species (ROS) that in turn drive the nuclear-to-cytoplasmic translocation of APE1, contributing to an APE1-mediated positive feedback effect. In a nude mouse model, LogiCas achieves tumor-site-specific drug release, demonstrating robust antitumor efficacy with ∼91% tumor inhibition, without obvious systemic toxicity. This work provides a new design strategy for integrating protein and nucleic acid biomarkers across molecular levels to enable CRISPR-mediated precision therapeutic activation.},
}

Animals
Humans
*MicroRNAs/genetics
Mice, Nude
*DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics/metabolism
Photochemotherapy/methods
*Neoplasms/therapy/genetics/drug therapy
Cell Line, Tumor
Precision Medicine
*CRISPR-Cas Systems
*Antineoplastic Agents/administration & dosage
Reactive Oxygen Species/metabolism
Drug Liberation
Mice
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42914884,
year = {2027},
author = {Wu, Z and Ma, Y and Li, X and Yan, Z and Huang, Y and Wang, K and Quan, W and Mo, L and Liu, SG},
title = {CRISPR/Cas12a-powered self-sustaining catalytic nucleic acid circuit for highly sensitive detection of aflatoxin B1.},
journal = {Talanta},
volume = {313},
number = {Pt B},
pages = {130809},
doi = {10.1016/j.talanta.2026.130809},
pmid = {42914884},
issn = {1873-3573},
mesh = {*Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Catalysis ; },
abstract = {Aflatoxin B1 (AFB1) is a highly toxic mycotoxin and strongly carcinogenic and teratogenic to humans. Achieving CRISPR-based sensing system for highly sensitive detection of AFB1 remains a significant challenge. A CRISPR/Cas12a-powered self-sustaining catalytic nucleic acid circuit (CPSCN) is proposed for the sensitive and accurate detection of aflatoxin B1 (AFB1). In the CPSCN sensing system, CRISPR/Cas12a/gRNA1 indirectly recognizes AFB1 by aptamer and effectively triggers trans-cleavage of a pair of self-reporting single-stranded DNA-locked RNA2 fluorescence probes (named as dual-winged fluorescence probe). The released RNA2 then initiates an autocatalytic positive feedback loop, resulting in continuous probe cleavage. The CPSCN system achieves highly sensitive detection in a single-enzyme one-step process. The method exhibits excellent quantitative capability for AFB1 detection with a detection limit of 7.5 pg/mL. In real samples, the detection results of the proposed biosensor show high consistency with those obtained by enzyme-linked immunosorbent assay (ELISA). This work not only provides a novel strategy for AFB1 detection with high sensitivity, robustness, and operational simplicity, but also offers a new insight for designing signal amplification strategies of CRISPR-based biosensors.},
}

*Aflatoxin B1/analysis
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Aptamers, Nucleotide/chemistry
Limit of Detection
Catalysis

@article {pmid42943590,
year = {2026},
author = {Li, S and Gao, J and Miao, Y and Chen, Q and Mu, H and Zheng, H and Zhang, Y and Zhou, J and Huang, C and Li, M and Yu, Y and Yang, L},
title = {A Review of Recombinase-Aided Amplification (RAA) in Veterinary Medicine.},
journal = {Microorganisms},
volume = {14},
number = {10},
pages = {},
pmid = {42943590},
issn = {2076-2607},
support = {25531C//Chongqing Academy of Animal Science/ ; 26519C//Municipal (Provincial-level) Financial Research Project of Chongqing Academy of Animal Sciences/ ; NCTIP-XD/B19//National Center for Technology Innovation for Pigs/ ; MARA-ORS261-AH024//Basic and Long Term Agricultural Science and Technology Observation and Research/ ; },
abstract = {Rapid and accurate pathogen detection is crucial to mitigating the significant economic impacts of infectious animal diseases. Recombinase-aided amplification (RAA) has emerged as a highly promising isothermal diagnostic tool, characterized by its rapid turnaround time, robust amplification efficiency, and single-copy analytical sensitivity. In veterinary diagnostics, RAA has evolved from single-pathogen proof-of-concept assays to become a promising technology for point-of-care scenarios. This review critically evaluates the integration of RAA with real-time fluorescence, CRISPR-Cas systems, lateral flow dipsticks (LFDs), and suspension microarray technologies, highlighting their impact on assay sensitivity and point-of-care (POC) applicability. Despite recent advancements, including encouraging progress in multiplexing and expanded applications in companion animal medicine, inherent challenges such as matrix effects in crude clinical samples, aerosol contamination risks, and commercialization barriers remain. By benchmarking RAA against traditional molecular methods and structurally similar technologies like recombinase polymerase amplification (RPA), this review outlines future trajectories for optimizing RAA platforms, aiming to maximize their utility in routine veterinary surveillance and epidemiological control.},
}

@article {pmid42943678,
year = {2026},
author = {Wang, Z and Wang, J and Li, L and Chen, G and Wen, H and Liu, B and Dong, Y},
title = {CRISPR/Cas12a-Based Aptasensor for Cas9 Protein Detection in Spiked Food Matrices.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {20},
pages = {},
pmid = {42943678},
issn = {1420-3049},
support = {GWKF2025-006//Key Laboratory of Quality and Safety of Cereals and Their Products, State Administration for Market Regulation；Quality and Safety Institute of Agricultural Products, Heilongjiang Academy of Agricultural Sciences/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Protein 9/analysis ; Molecular Docking Simulation ; *Food Analysis/methods ; },
abstract = {Residual gene editors are a concern associated with the widespread application of CRISPR/Cas9 technology. Here, we developed a CRISPR/Cas12a-based fluorescent aptasensor for the qualitative and quantitative detection of Cas9 protein. Secondary structure prediction and molecular docking were combined to identify potential binding sites of the Cas9-specific aptamer. A fluorescence aptasensor was constructed based on competitive binding: in the presence of Cas9, the aptamer binds to the target and releases complementary DNA (cDNA), which activates the trans-cleavage activity of Cas12a. Cas12a subsequently cleaves a single-stranded DNA (ssDNA) reporter labeled with a fluorophore and a quencher, generating a fluorescence signal, expressed in relative fluorescence units (RFU), that increases with Cas9 concentration. The optimized conditions comprised a trans-cleavage reaction time of 20 min, a Cas12a:CRISPR RNA (crRNA) molar ratio of 1:1, a final aptamer concentration of 180 nM, and a combination of low- and high-salt buffers to balance molecular recognition and protein stability. The proposed assay enabled Cas9 protein detection in buffer and Cas9-spiked soybean milk and cereal matrices. These results represent a proof-of-concept evaluation in complex food matrices rather than validation of authentic residual Cas9 detection. Further studies using unspiked gene-edited samples with independently verified Cas9 residues and an orthogonal reference method are required.},
}

*CRISPR-Cas Systems/genetics
*Aptamers, Nucleotide/chemistry/genetics
*Biosensing Techniques/methods
*CRISPR-Associated Protein 9/analysis
Molecular Docking Simulation
*Food Analysis/methods

@article {pmid42505080,
year = {2026},
author = {Yin, J and Gao, Y and Jing, Y and Jiang, X and Wang, F and Cai, Y and Li, MH and Cao, T and Wang, M and He, Y and Zhao, H and Zhang, S and Ma, S and Wang, S and Liu, GH and Zhang, W and Qu, J},
title = {CRISPR screening identifies GNPTAB as a noncanonical STING activator driving cellular senescence.},
journal = {Protein & cell},
volume = {17},
number = {11},
pages = {1046-1059},
pmid = {42505080},
issn = {1674-8018},
support = {2024ZD0530400//Chronic Diseases-National Science and Technology Major Project/ ; 2022YFA1103700//National Key Research and Development Program of China/ ; 2025YFA1805300//National Key Research and Development Program of China/ ; 82488301//National Natural Science Foundation of China/ ; 82125011//National Natural Science Foundation of China/ ; 82322025//National Natural Science Foundation of China/ ; 82330044//National Natural Science Foundation of China/ ; 32341001//National Natural Science Foundation of China/ ; 82361148131//National Natural Science Foundation of China/ ; 82361148130//National Natural Science Foundation of China/ ; 82350710800//National Natural Science Foundation of China/ ; 32121001//National Natural Science Foundation of China/ ; 82192863//National Natural Science Foundation of China/ ; 82471586//National Natural Science Foundation of China/ ; 92468303//National Natural Science Foundation of China/ ; 82401822//National Natural Science Foundation of China/ ; 82271600//National Natural Science Foundation of China/ ; Z240018//Program of the Beijing Natural Science Foundation/ ; F251011//Program of the Beijing Natural Science Foundation/ ; Z230011//Program of the Beijing Natural Science Foundation/ ; JQ24044//Program of the Beijing Natural Science Foundation/ ; C2406001//Shenzhen Medical Research Fund/ ; B2504003//Shenzhen Medical Research Fund/ ; YSBR-076//CAS Project for Young Scientists in Basic Research/ ; YSBR-012//CAS Project for Young Scientists in Basic Research/ ; 20250484873//Beijing Nova Program/ ; 2024AZB3002//Beijing Anzhen Hospital High Level Research Funding/ ; //New Cornerstone Science Foundation through the XPLORER PRIZE/ ; XDC0200000//CAS Youth Interdisciplinary Team, the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; XDA0460403-05//CAS Youth Interdisciplinary Team, the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; ZJAD-2024001//Key Laboratory of Alzheimer's Disease of Zhejiang Province/ ; YESS20240793//Young Elite Scientists Sponsorship Program by CAST/ ; 2023IOZ0202//Initiative Scientific Research Program, Institute of Zoology, CAS/ ; 2024IOZ0103//Initiative Scientific Research Program, Institute of Zoology, CAS/ ; 2023IOZ0102//Initiative Scientific Research Program, Institute of Zoology, CAS/ ; 825QN316//Hainan Provincial Natural Science Foundation of China/ ; 826MS0172//Hainan Provincial Natural Science Foundation of China/ ; 2023ICAC-YANFA//Key Research and Development Project of Hainan Province/ ; RP2500000375//HNMU Start-up Package-ICAC/ ; RP2400000981//HNMU Start-up Package-ICAC/ ; HYZHXMH01012//Space Medical Experiment Project of CMSP/ ; },
mesh = {Humans ; STING Protein ; *Cellular Senescence/genetics ; *Membrane Proteins/metabolism/genetics ; Lysosomes/metabolism ; cGAS-STING Signaling Pathway ; *Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Protein Serine-Threonine Kinases/metabolism/genetics ; Signal Transduction ; },
abstract = {Cellular senescence is accompanied by profound lysosomal alterations, yet whether lysosome-associated factors actively drive aging remains unclear. Through a focused CRISPR/Cas9 screen in human mesenchymal progenitor cells (hMPCs), we identified N-acetylglucosamine-1-phosphotransferase subunits alpha and beta (GNPTAB), an enzyme responsible for lysosomal hydrolase targeting, as a positive regulator of cellular senescence. Genetic ablation of GNPTAB attenuated senescence, whereas its overexpression accelerated senescence. This pro-senescent function occurred independently of GNPTAB's canonical enzymatic role. Instead, GNPTAB binds to the innate immune adaptor stimulator of interferon genes (STING) through an interface involving the critical residue E1119, leading to activation of STING and its downstream TANK-binding kinase 1 (TBK1), as well as pro-inflammatory gene expression. A STING-binding-deficient GNPTAB mutant (E1119A) preserved canonical lysosomal functions but failed to induce senescence, while STING depletion abolished GNPTAB-driven senescence. Together, these findings uncover a new signaling pathway wherein GNPTAB engages STING to facilitate its activation, suggesting that the GNPTAB-STING interaction interface may serve as a potential therapeutic target for ameliorating age-related cellular dysfunction.},
}

Humans
STING Protein
*Cellular Senescence/genetics
*Membrane Proteins/metabolism/genetics
Lysosomes/metabolism
cGAS-STING Signaling Pathway
*Mesenchymal Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
Protein Serine-Threonine Kinases/metabolism/genetics
Signal Transduction

@article {pmid42749195,
year = {2027},
author = {Greeshma, M and Bhat, AI},
title = {CRISPR-Cas12a-assisted RT-RPA-LFA for rapid cluster IV-specific detection of cardamom mosaic virus in crude plant extracts.},
journal = {Journal of virological methods},
volume = {347},
number = {},
pages = {115468},
doi = {10.1016/j.jviromet.2026.115468},
pmid = {42749195},
issn = {1879-0984},
mesh = {Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Plant Diseases/virology ; *Plant Extracts ; *CRISPR-Cas Systems ; *Begomovirus/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; },
abstract = {Cardamom mosaic virus (CdMV), associated with mosaic/katte disease in cardamom, is a highly destructive virus that leads to severe reductions in plant growth and yield. High levels of genetic variability approaching species-level divergence exist among CdMV isolates from different geographical regions. Development of a cluster-specific diagnostic assay can enable reliable detection of locally prevalent virus populations and differentiate them from divergent ones. Therefore, in the present study, a CRISPR-Cas12a-coupled reverse transcription-recombinase polymerase amplification-lateral flow assay (RT-RPA-CRISPR-LFA) was developed for cluster IV-specific detection of Idukki isolates of CdMV. The viral target sequence (coat protein) was first amplified through RT-RPA using virus-specific primers designed from conserved region present in all strains of CdMV and crude plant extract template. The amplified product was subsequently recognized by CRISPR-Cas12a complex guided by a crRNA designed to specifically detect CdMV isolates associated with Idukki region of Kerala, triggering trans-cleavage activity of the complex and subsequent cleavage of the lateral flow reporter (LF reporter). This generated a visually detectable signal by LFA. The optimum assay performance was achieved with an RT-RPA incubation time of 10 min, LF reporter, Cas12a, and crRNA concentrations of 500, 75, and 125nM, respectively, and a CRISPR-Cas12a reaction incubation time of 1h. The assay was specific for CdMV strains representing Idukki region of Kerala and exhibited a detection limit of 1 fg (2.4 × 10[2] copies), which was 10 times less sensitive than qPCR. Practical feasibility and consistency of the assay were verified through validation using different field samples of cardamom.},
}

Sensitivity and Specificity
Rapid Diagnostic Tests
*Plant Diseases/virology
*Plant Extracts
*CRISPR-Cas Systems
*Begomovirus/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
DNA Primers/genetics

@article {pmid42868209,
year = {2026},
author = {Lahlou, G and Cornille, M and Lai, N and Lecomte, MJ and Safieddine, S},
title = {Emerging Therapies for Genetic Deafness: The Rise of Precision Gene Editing.},
journal = {Ear and hearing},
volume = {47},
number = {6},
pages = {1497-1508},
pmid = {42868209},
issn = {1538-4667},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *Deafness/genetics/therapy ; Mice ; Disease Models, Animal ; CRISPR-Cas Systems ; Genetic Vectors ; },
abstract = {Hearing loss is a prevalent sensory disorder with profound social and health impacts. Although hearing aids and cochlear implants can significantly restore auditory perception, they do not address the underlying genetic causes. Inherited forms of hearing loss, often monogenic, are ideal candidates for gene therapy, particularly since over 150 genes have been linked to deafness. Advances in vector engineering and genome editing technologies now enable precise targeting of auditory cells within the inner ear. Technologies such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas systems, base editing, and prime editing offer powerful tools to correct pathogenic variants at their native genomic loci. This review summarizes the current landscape of gene editing approaches for both hereditary and acquired hearing loss, emphasizing key preclinical studies in mouse models encompassing dominant and recessive forms of deafness. We discuss the advantages and limitations of different editing techniques, vector delivery systems, and administration routes, and explore obstacles including editing efficiency, cell-specific targeting, and the translation of these therapies to humans. Collectively, these advances represent a pivotal step toward variant-specific, durable, and potentially curative treatments for hearing loss.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
Animals
*Deafness/genetics/therapy
Mice
Disease Models, Animal
CRISPR-Cas Systems
Genetic Vectors

@article {pmid42936269,
year = {2026},
author = {Yin, Q and Chen, L},
title = {xNNPCD identifies regulators of programmed cell death by integrating perturbation transcriptomes with cancer dependency profiles.},
journal = {NAR cancer},
volume = {8},
number = {4},
pages = {zcag031},
pmid = {42936269},
issn = {2632-8674},
support = {R01 CA301708/CA/NCI NIH HHS/United States ; R01 NS139485/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/genetics/pathology/drug therapy ; *Apoptosis/genetics/drug effects ; *Transcriptome ; Endoplasmic Reticulum Chaperone BiP ; Gene Expression Regulation, Neoplastic ; *Computational Biology/methods ; CRISPR-Cas Systems ; Ferroptosis/drug effects/genetics ; },
abstract = {Programmed cell death (PCD) encompasses regulated processes whose dysregulation shapes cancer fitness, yet computational studies use known PCD genes for prognosis rather than discovering regulators. We developed xNNPCD, an interpretable neural-network framework that links CRISPR-Cas9 perturbation signatures from CMap to gene dependency profiles from DepMap. The model constrains hidden neurons to five PCD pathways and iteratively refines a prior gene-PCD pathway mask matrix derived from GO, KEGG, and Reactome using pathway-neuron ablation. This converts binary gene-pathway relationships into continuous-valued associations and improves dependency prediction over random forests, fully connected multi-layer perceptrons, and its own non-iterative variant. The learned mask matrix recovers annotated death regulators and nominates candidate regulators, including RPL23A, HSPA5, SNRPA1, SLC6A2, and ASAH1; combined with dependency scores, it separates pathway coupling from regulatory direction. Transferring the refined mask matrix and learned weights to compound-induced perturbation data enables in silico drug screening, identifying BRD-K19103580 and decitabine as therapeutic agents for apoptosis and ferroptosis, respectively. The pathway-resolved drug profiles can facilitate the design of combination therapies targeting complementary PCD pathways to overcome single-pathway resistance. Overall, xNNPCD offers a pan-cancer, interpretable approach that generalizes across perturbation modalities for mapping the regulatory landscape and elucidating the molecular processes of PCD in cancer.},
}

Humans
*Neoplasms/genetics/pathology/drug therapy
*Apoptosis/genetics/drug effects
*Transcriptome
Endoplasmic Reticulum Chaperone BiP
Gene Expression Regulation, Neoplastic
*Computational Biology/methods
CRISPR-Cas Systems
Ferroptosis/drug effects/genetics

@article {pmid42937587,
year = {2026},
author = {Wang, Y and Huang, E and Zhang, Z and Zhu, X and Zhang, T and Yang, N and Chen, Z and Cai, D and Liu, D},
title = {Fluorescence Encoding-Enabled Multiplexed Droplet Digital Recombinase Polymerase Amplification-CRISPR/Cas12a Assay for Rapid Screening of Lower Respiratory Tract Infection-Associated Bacterial Pathogens.},
journal = {ACS nano},
volume = {20},
number = {42},
pages = {28655-28667},
doi = {10.1021/acsnano.6c13618},
pmid = {42937587},
issn = {1936-086X},
support = {2024A1515011810//Natural Science Foundation of Guangdong Province/ ; 2024A1515220086//Natural Science Foundation of Guangdong Province/ ; 82372345//National Natural Science Foundation of China (NSFC)/ ; 82472372//National Natural Science Foundation of China (NSFC)/ ; 82572669//National Natural Science Foundation of China (NSFC)/ ; HM2025002//Clinical Research Hongmian Project of Guangzhou First People?s Hospital/ ; PT82372345//Science Foundation of Guangzhou First People?s Hospital/ ; PT82472372//Science Foundation of Guangzhou First People?s Hospital/ ; PT82572669//Science Foundation of Guangzhou First People?s Hospital/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Fluorescence ; *Respiratory Tract Infections/microbiology/diagnosis ; Humans ; CRISPR-Associated Proteins/genetics ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Digital RPA-CRISPR/Cas12a assays enable accurate nucleic acid quantification; however, their multiplexing capacity is inherently limited by signal confounding arising from the promiscuous trans-cleavage activity of Cas12a effectors. To overcome this limitation, we developed a fluorescence encoding-enabled multiplexed droplet digital RPA-CRISPR/Cas12a (FLEMD-CRISPR) assay that integrates combinatorial fluorescence encoding, deterministic droplet pairing within a calabash-shaped microwell array, and a machine learning-assisted decoding pipeline. In FLEMD-CRISPR, small-volume fluorescence-encoded CRISPR/Cas12a droplets, each assigned a unique spectral signature corresponding to a specific crRNA, are one-to-one paired with large-volume RPA droplets. Following RPA amplification and droplet merging, Cas12a-mediated reporter cleavage occurs only in droplets containing target amplicons. The decoding pipeline then localizes droplets, classifies their colors to assign crRNA identities, and finally detects positive signals, enabling high-throughput, hands-off analysis of the entire droplet array. FLEMD-CRISPR enabled simultaneous quantification of nucleic acid targets from eight major lower respiratory tract infection-associated bacterial pathogens, showing strong linear correlation (R2 = 0.9987-0.9998) and consistent detection efficiency (slopes = 0.9849-1.0198) relative to single-plex digital PCR. The complete workflow, from sample preparation to final readout, can be completed within 2 h. Clinical validation using 30 bronchoalveolar lavage fluid samples showed that the bacterial detection outcomes of FLEMD-CRISPR were highly concordant with standard-of-care culture, achieving 100% (20/20) positive percent agreement and 99.1% (218/220) negative percent agreement. By enabling panel reconfiguration through simple modifications of the droplet encoding library, FLEMD-CRISPR provides a flexible platform for the development of highly multiplexed digital CRISPR assays.},
}

*CRISPR-Cas Systems/genetics
Fluorescence
*Respiratory Tract Infections/microbiology/diagnosis
Humans
CRISPR-Associated Proteins/genetics
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods

@article {pmid42938263,
year = {2026},
author = {Alsaady, IM},
title = {CRISPR/Cas-Based Molecular Diagnostics for Intestinal Parasites: From Proof-of-Concept to Point-of-Care Testing.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {16},
number = {20},
pages = {},
pmid = {42938263},
issn = {2075-4418},
abstract = {Intestinal parasitic infections remain a significant global public health concern, particularly in low- and middle-income countries, where timely and accurate diagnosis is essential for effective disease management and control. Although conventional diagnostic methods, including microscopy, antigen detection, and polymerase chain reaction (PCR), are widely used, each possesses inherent limitations related to sensitivity, turnaround time, laboratory infrastructure, or point-of-care applicability. Recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems have led to the development of highly sensitive and rapid molecular diagnostic platforms suitable for decentralized testing. This review summarizes current applications of CRISPR/Cas-based diagnostics for intestinal and foodborne parasitic infections, with particular emphasis on assays developed for Giardia duodenalis, Cryptosporidium parvum, Clonorchis sinensis, Fasciola hepatica, and Schistosoma spp. Published studies have predominantly employed Cas12a, with additional applications of Cas12b and Cas13a and, more recently, a type I-A Cascade-Cas3 system, generally coupled with isothermal amplification methods such as recombinase polymerase amplification (RPA) or recombinase-aided amplification (RAA), enabling highly sensitive detection within 60-90 min using fluorescence or lateral-flow strip readouts. The available evidence indicates high analytical sensitivity and specificity, with several assays demonstrating substantial agreement with conventional molecular methods and potential for point-of-care development. However, current CRISPR-based assays remain limited by dependence on nucleic acid extraction, relatively small clinical validation cohorts, lack of standardized protocols, and the persistence of false-negative results associated with low parasite burdens and pre-analytical variables. Emerging strategies, including simplified or extraction-free sample preparation, one-pot sample-to-answer platforms, and detection of parasite-derived cell-free DNA, may further enhance the clinical utility of CRISPR diagnostics. Overall, CRISPR/Cas-based technologies represent a promising next-generation approach for intestinal parasite diagnosis, although large-scale clinical validation and workflow optimization are still required before widespread implementation in routine clinical and field settings.},
}

@article {pmid42938363,
year = {2026},
author = {Aliev, T and Imatdinov, A and Prudnikova, E and Imatdinov, I},
title = {Engineering of the High-Precision Cas12m Dual Base Editor-Loaded Virus-like Particles for A-to-G and C-to-T Conversions.},
journal = {Genes},
volume = {17},
number = {10},
pages = {},
pmid = {42938363},
issn = {2073-4425},
support = {The Federal Scientific-technical programme for genetic technologies development for 2019-2030, agreement № 075-15-2025-526//the Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; *Virion/genetics ; *Bacterial Proteins/genetics ; },
abstract = {Background/Objectives: CRISPR/Cas-dependent base editing enables the precise correction of genomic sequences by direct modification of DNA bases without creating potentially mutagenic DNA double-strand breaks. Base editing is considered a therapeutic approach for rare monogenic disorders or viral infections. However, many variants of base editors suffer from limited specificity because they induce bystander edits within a broad editing window. However, nucleotide substitution is sufficient to correct a mutation or to achieve a therapeutic effect. Methods: PCR, cloning by restriction digest, and ligation were used to create genetic constructs. The editing efficiency was evaluated using NGS. Virus-like particles were produced in HEK293T cells using transient expression. VLPs were visualized by electron microscopy. Results: Using dual base editors that induce C-to-T and A-to-G substitutions, we show that high editing precision can be achieved by engineering the linker between the MmCas12m domain and the deaminase domain of the editor. By testing different linker sequences, we have created high-precision dual base editors with narrowed editing windows that can edit a single nucleotide at a target position with high accuracy. Conclusions: We found that shortening the linker to an acceptable length provides a single-nucleotide substitution in vitro with minimal bystander activity (less than 0.8%), while the high-precision MmCas12m dual base editor retains up to 90% of the editing efficiency of the original base editor. These high-precision base editors will be useful in cases where high precision is required, and editing of adjacent nucleotides is undesirable.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
HEK293 Cells
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics
*Virion/genetics
*Bacterial Proteins/genetics

@article {pmid42941827,
year = {2026},
author = {Duan, Y and Xu, K and Zhou, H and He, C and Ding, J and Qin, C and Zhang, Y and Han, C and Meng, H},
title = {IndelQuant: A Workflow for Cost-Efficient Amplicon Sequencing Analysis of Genome-Edited Chickens.},
journal = {International journal of molecular sciences},
volume = {27},
number = {20},
pages = {},
pmid = {42941827},
issn = {1422-0067},
support = {2024-02-08-00-12-F00052//Shanghai Municipal Agricultural Commission/ ; },
mesh = {Animals ; *Chickens/genetics ; *INDEL Mutation ; *Gene Editing/methods/economics ; CRISPR-Cas Systems ; Genome ; Workflow ; *High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; Cost-Benefit Analysis ; },
abstract = {Accurate quantification of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-induced insertions and deletions (indels) is essential in chicken genome-editing studies, where low editing efficiencies and mosaicism can complicate the assessment of target-site modifications. Existing assays often involve a trade-off between affordability and the sensitivity orsequence information needed for detailed analysis. Here, we introduce IndelQuant, a cost-efficient workflow integrating sample-specific barcode PCR, pooled amplicon library construction, AdapterRemoval3-based read processing, and CRISPResso2-based indel quantification. The workflow provides graphical and tabularsummaries of editing efficiency, indel positions, indel-size distributions, and sample-level mutation profiles. We evaluated the workflow in TYR-edited DF-1 cells and genome-edited chickens. In edited chickens, IndelQuant detected tissue-specific indel signals, most below 5%, while negative controls showed background frequencies from 0 to 0.03%. These findings indicate that IndelQuant retains the sequence-level information provided by amplicon sequencing while simplifying sample processing and analysis, offering a practical approach to routine screening for low-frequency and mosaic indels in poultry genome-editing studies.},
}

Animals
*Chickens/genetics
*INDEL Mutation
*Gene Editing/methods/economics
CRISPR-Cas Systems
Genome
Workflow
*High-Throughput Nucleotide Sequencing/methods
Sequence Analysis, DNA/methods
Cost-Benefit Analysis

@article {pmid42942050,
year = {2026},
author = {Irum, and Tripathi, G and Ahmad, S and Fatima, N and Puri, P and Ahmad, N and Dissanayake, KK and Ahmad, K and Ahmad, S and Farooqui, A},
title = {Terpenoids: A Comprehensive Review on Biosynthesis, Applications and Enhancement Strategies for Translational Research.},
journal = {International journal of molecular sciences},
volume = {27},
number = {20},
pages = {},
pmid = {42942050},
issn = {1422-0067},
support = {(QU-APC-2026)//Qassim University/ ; },
mesh = {*Terpenes/metabolism/chemistry/therapeutic use/pharmacology ; Humans ; Animals ; *Translational Research, Biomedical ; Metabolic Engineering/methods ; Biological Products/chemistry/therapeutic use/metabolism ; },
abstract = {There is growing interest in natural bioactive compounds as potentially safer and more sustainable sources of therapeutic agents. Terpenoids, one of the most structurally diverse and biologically active classes of natural products, are derived from repeating isoprene units and possess a broad range of pharmacological and industrial applications. This review provides a comprehensive overview of recent advances in terpenoid biosynthesis, structural classification, and therapeutic applications. It also discusses emerging approaches for enhancing large-scale production, improving functional efficacy, and overcoming pharmacokinetic limitations. Attention is given to terpenoids currently undergoing clinical evaluation, along with their progress toward commercial development. Despite substantial preclinical evidence supporting their therapeutic potential, the clinical translation of many terpenoids remains limited. Key challenges include poor aqueous solubility, low permeability, slow dissolution, and high lipophilicity, which can collectively restrict systemic bioavailability and therapeutic efficacy. Moreover, only a relatively small proportion of biologically active terpenoids have progressed to advanced stages of clinical development, highlighting a considerable gap between experimental findings and clinical application. This review integrates current knowledge of terpenoid biosynthesis, biological activities, production strategies, and translational development while highlighting sustainable and scientifically driven approaches that may facilitate their future therapeutic and commercial utilization.},
}

*Terpenes/metabolism/chemistry/therapeutic use/pharmacology
Humans
Animals
*Translational Research, Biomedical
Metabolic Engineering/methods
Biological Products/chemistry/therapeutic use/metabolism

@article {pmid42942146,
year = {2026},
author = {Dildabayeva, D and Samarkhanova, D and Lezina, L},
title = {Beyond Homologous Recombination: Functional Modules of Olaparib Resistance Revealed by CRISPR Screens.},
journal = {International journal of molecular sciences},
volume = {27},
number = {20},
pages = {},
pmid = {42942146},
issn = {1422-0067},
support = {AP234846678//Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Phthalazines/pharmacology/therapeutic use ; *Piperazines/pharmacology/therapeutic use ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Homologous Recombination ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *CRISPR-Cas Systems ; Animals ; DNA Repair ; },
abstract = {Clinical use of PARP inhibitors (PARPi), including olaparib, has improved outcomes for patients with BRCA1/2-mutant and homologous recombination (HR)-deficient tumors. However, acquired resistance remains a major limitation to their efficacy. CRISPR-Cas9 screening approaches have been developed to identify genetic drivers of PARPi resistance. However, differences in experimental conditions, library coverage, and selection design complicate comparisons across studies. In this review, we present a harmonized reanalysis of CRISPR knockout olaparib resistance screens to identify recurrent resistance-associated genes and pathways. Datasets with raw data were collected from the BioGRID ORCS database. Resistance hits identified in at least two screens were subjected to Reactome pathway-enrichment analysis and organized into functional modules. Using this approach, we identified eight modules: DNA-damage repair and replication stress; cell-cycle regulation; chromatin remodeling and histone modification; post-translational regulation; mitochondrial function; TGF-β and mTOR signaling; RNA processing and nucleocytoplasmic transport; and emerging adaptive mechanisms. Importantly, mitochondrial function genes were observed as recurrent hits, but their relation to olaparib resistance is underinvestigated. Our integrative analysis suggests that PARPi resistance is a complex, adaptive phenotype shaped by recurrent but context-dependent vulnerabilities across multiple cellular programs. This framework prioritizes candidate resistance drivers for validation and highlights directions for the design of combination therapies aimed at overcoming PARPi resistance.},
}

*Phthalazines/pharmacology/therapeutic use
*Piperazines/pharmacology/therapeutic use
Humans
*Drug Resistance, Neoplasm/genetics
*Homologous Recombination
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
*CRISPR-Cas Systems
Animals
DNA Repair

@article {pmid42943380,
year = {2026},
author = {Gao, Y and Dong, H and Fang, Z and Liu, S and Xu, Y and Guo, Y and Yang, Y and Luo, J and Yan, L},
title = {Regulatory Pathways of Endophytic Fungi in Promoting Flavonoid Biosynthesis in Medicinal Plants.},
journal = {Microorganisms},
volume = {14},
number = {10},
pages = {},
pmid = {42943380},
issn = {2076-2607},
support = {PL2025H203//Heilongjiang Provincial Natural Science Foundation/ ; YQJH2025093//Young Talents of Basic Research in Universities of Heilongjiang Province/ ; 2025-KYYWF-ZR0104//Fundamental Research Funds in Universities of Heilongjiang Province/ ; ZHY2025-084//Heilongjiang Provincial Traditional Chinese Medicine Research Project/ ; },
abstract = {Endophytic fungi form complex symbiotic associations with medicinal plants via long-term coevolution. Such cross-kingdom interactions substantially modulate flavonoid biosynthesis in host plants. This review dissects the mechanisms through which endophytic fungi elevate flavonoid accumulation in medicinal plants. We first summarize symbiotic and growth-promoting relationships between endophytic fungi and medicinal plants, outlining four core synergistic functions: nutrient supply, phytohormone homeostasis, stress resistance enhancement, and secondary metabolism activation. We then elaborate on three primary regulatory patterns underlying endophyte-modulated flavonoid biosynthesis, including direct activation of plant flavonoid biosynthetic pathways, indirect regulation via stress mitigation and host metabolic reprogramming, and autonomous flavonoid production by endophytic fungi. Notably, most current evidence for autonomous flavonoid biosynthesis by endophytic fungi is derived from axenic cultures, whereas its occurrence and quantitative contribution within colonized plant tissues remain to be established. We further discuss state-of-the-art technologies for untangling plant-fungus symbiotic interactions, including multi-omics integration, spatial metabolomics, single-cell sequencing, synthetic microbial consortia, and CRISPR-Cas genome editing. Lastly, we propose future research perspectives covering green cultivation of medicinal plants, targeted improvement of medicinal quality, and efficient microbial biosynthesis of flavonoids.},
}

@article {pmid42943509,
year = {2026},
author = {Aristimuño Ficoseco, CM and Carrasco, N and Montemurro, M and Chieffi, D and Fadda, SG and Nader-Macias, MEF and Fusco, V and Vignolo, GM and Fanelli, F},
title = {Genomic Characterization of the Probiotic Limosilactobacillus mucosae CRL2069 Isolated from Feedlot Cattle Feces.},
journal = {Microorganisms},
volume = {14},
number = {10},
pages = {},
pmid = {42943509},
issn = {2076-2607},
support = {//Joint Bilateral Agreement ONK/National Scientific and Technical Research council (Argentina)/ ; },
abstract = {Limosilactobacillus mucosae (L.) CRL2069, isolated from feedlot cattle feces, has previously exhibited high performance when used as a probiotic in animals. To investigate the genetic bases underlying its probiotic potential, complete genome sequencing and comparative analyses were performed. The CRL2069 genome showed a total length of 1.93 Mb, with an average GC content of 46.76%. No evidence of plasmid sequences was detected, while CRISPR-Cas elements, as well as twelve genomic islands, were identified in the genome. Pan-genomic and clustering analysis among L. mucosae strains showed a lack of partitioning according to their isolation origin, revealing a niche specialization. CRL2069 genes related to stress and those conferring probiotic character were identified. In addition, the absence of acquired antimicrobial resistance genes and virulence determinants evidenced the safety of this strain. Genomic determinants conferring the ability to metabolize a diverse array of carbohydrates, particularly those present in cattle feed, were annotated. Phenotypic analysis supported the genomic prediction and demonstrated the capacity of CRL2069 to metabolize 39 out of 95 carbon sources tested and to produce folate. Based on the results obtained, L. mucosae CRL2069 evaluated in this study is confirmed to be a promising candidate for use as a probiotic in cattle.},
}

@article {pmid42501539,
year = {2026},
author = {Qin, C and Jiang, S and Xiao, F and Huang, J and Jin, M and Yang, K and Meng, H},
title = {DAZL-targeted inducible kill switches enable efficient ablation of chicken primordial germ cells.},
journal = {Poultry science},
volume = {105},
number = {11},
pages = {107459},
pmid = {42501539},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Germ Cells ; *RNA-Binding Proteins/genetics/metabolism ; *Avian Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Chick Embryo ; *Genes, Transgenic, Suicide ; },
abstract = {Competition from endogenous primordial germ cells (PGCs) in recipient embryos limits the generation of fully donor-derived offspring in avian surrogate-host systems. An inducible and germline-restricted ablation strategy is therefore needed to create sterile recipients without compromising somatic development. Using CRISPR/Cas9-mediated homology-directed repair, we inserted three inducible suicide-gene cassettes (iCaspase9, RapaCasp9, and CD) into the endogenous DAZL locus of chicken PGCs. All knock-in lines showed stable reporter expression and retained typical PGC morphology. Comparative functional analyses identified iCaspase9 activated by AP20187 as the most sensitive and specific ablation system, achieving near-complete killing at nanomolar concentrations without detectable toxicity in control cells. RapaCasp9 induced with rapamycin also ablated engineered PGCs efficiently, but rapamycin caused marked non-specific growth inhibition in control cells. Replacing rapamycin with the synthetic A/C heterodimerizer AP21967 largely eliminated this off-target toxicity while preserving rapid and robust killing. By contrast, yeast-derived CD variants did not substantially improve 5-fluorocytosine sensitivity in chicken PGCs. DAZL-restricted iCaspase9/AP20187 and the optimized RapaCasp9/AP21967 system provide efficient inducible kill switches for chicken PGCs in vitro. These platforms establish a practical genetic toolkit for generating sterile surrogate hosts to support avian genome editing, germline replacement, and Sire Dam Surrogate breeding.},
}

Animals
*Chickens/genetics
*Germ Cells
*RNA-Binding Proteins/genetics/metabolism
*Avian Proteins/genetics/metabolism
CRISPR-Cas Systems
Chick Embryo
*Genes, Transgenic, Suicide

@article {pmid42580267,
year = {2026},
author = {Farooq, M and Han, D and Zhang, L and Wadood, AA and Geng, Q and Chen, J and Liang, K and Xi, Y and Jiao, X and Hou, J and Zhang, Y},
title = {Clustered regularly interspaced short palindromic repeats‑associated protein 9 (CRISPR-Cas9) based genome editing in avian primordial germ cells: Comparative technologies, translational applications, and regulatory challenges.},
journal = {Poultry science},
volume = {105},
number = {11},
pages = {107469},
pmid = {42580267},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Germ Cells ; *Chickens/genetics ; },
abstract = {Primordial germ cells (PGCs) are a unique platform for heritable gene editing in avian species, because they allow easy isolation, culture, and can then be reintroduced into the host. CRISPR/Cas9 technologies have advanced avian genome editing by enabling targeted editing of genes and traits for production, health, reproduction, and welfare. This review critically evaluates genome-editing tools in avian PGCs, including CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), base editing, and prime editing. Germline transmission efficiency, suitability, precision, and heritability are compared. Efficiency, cytotoxicity, and translational feasibility of delivery strategies, including viral vectors, electroporation and the use of ribonucleoproteins are assessed. Applications of these techniques in chickens are for muscle growth via myostatin (MSTN) gene disruption, viral resistance via editing of the sodium/hydrogen exchanger 1 (NHE1) gene, and control of male and female ratios through modification of sex determination genes. Additionally, applications in biopharmaceutical protein production and animal biodiversity conservation are also explored. Despite advances, some limitations remain, including low efficiency of homology-directed repair, off-target effects, mosaicism, and variability in transmission through the germ line. Current evidence demonstrates a significant lack of germline validation and scalability creating barriers to translate this potential into commercial avian production, especially, poultry breeding. Regulatory frameworks and their implications for food and commercialization are also discussed. Future research should prioritize precision editing, scalable delivery systems, and regulatory alignment to enable practical, ethical, and responsible implementation.},
}

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

@article {pmid42671510,
year = {2026},
author = {Roman, G and Lauritzen, KH and Smolkova, B and Almaas, R and Sandset, PM and Stavik, B and Sullivan, GJ and Chollet, ME},
title = {From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {8},
pages = {3790-3816},
pmid = {42671510},
issn = {2629-3277},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Ribonucleoproteins/metabolism/genetics ; *Single-Cell Analysis/methods ; },
abstract = {Precise genome editing of induced pluripotent stem cells (iPSCs) using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has opened unprecedented avenues for advancements in regenerative medicine and disease modelling. However, the establishment of isogenic single-cell-derived iPSC populations, particularly upon CRISPR-Cas9 gene editing modifications, is one of the major challenges still associated with these advanced methodologies relying on low-rate editing events and requiring defined clonogenicity. In response, we have developed a systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing. In particular, the protocol incorporates optimized single-cell cloning procedures for two commercially available dispensing platforms, one based on microfluidic imaging and the other on impedance technology, together with rapid droplet digital PCR (ddPCR)-based screening of non-homologous end joining (NHEJ) and homology-directed repair (HDR) outcomes. By combining gentle single-cell handling with advanced genotyping methodologies, the protocol enables efficient early assessment of editing outcomes before commitment to labour-intensive clonal derivation, thereby accelerating project timelines, minimising cell stress and loss, preserving genetic fidelity and supporting scalability. Coupled with precisely defined culture conditions tailored for post-seeding recovery, these approaches aim to improve iPSC viability and clonal outgrowth, achieving at least 60% in 96-well plate format within 10 days. Importantly, the protocol goes beyond step-by-step experimental instructions by providing comprehensive design strategies, decision-making criteria, and practical advice for avoiding and addressing the common pitfalls and unintended consequences of these advanced methods. Collectively, this integrated end-to-end approach provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Ribonucleoproteins/metabolism/genetics
*Single-Cell Analysis/methods

@article {pmid42700775,
year = {2027},
author = {Manjunatha Reddy, GB and Vignesh, M and Megha, NV and Brindhaa, R and Uzma, J and Shivasharanappa, N},
title = {Rapid point-of-care lateral flow assay for differentiation of capripoxviruses using RPA-CRISPR-Cas12a.},
journal = {Journal of virological methods},
volume = {347},
number = {},
pages = {115465},
doi = {10.1016/j.jviromet.2026.115465},
pmid = {42700775},
issn = {1879-0984},
mesh = {Animals ; *Capripoxvirus/isolation & purification/genetics/classification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Rapid Diagnostic Tests ; *Poxviridae Infections/diagnosis/veterinary/virology ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Cattle ; Goats ; Sheep ; Goat Diseases/diagnosis/virology ; Sheep Diseases/diagnosis/virology ; },
abstract = {The Capripoxviruses (CaPVs) are highly contagious viruses that includes sheeppox virus, goatpox virus, and lumpy skin disease virus infecting sheep, goats, and cattle respectively resulting in significant morbidity and mortality. This study presents a diagnostic tool based on recombinase polymerase amplification (RPA) assisted with CRISPR-Cas12a platform coupled with a lateral flow assay (LFA) that facilitate timely detection and differentiation of Capripox viruses, and support effective surveillance programs. The assay operates through the cleavage of a dual labelled oligonucleotide reporter molecule (Biotin-Fam), generating a detectable signal. The results can be interpreted either using a fluorescence-based detection system or through a lateral flow assay (LFA), where gold nanoparticles conjugated with anti-FAM antibodies enable visual signal detection. Under optimized assay conditions (50 nM LbCas12a and 1 µM reporter probe with a reaction time of 25 min), the CRISPR-Cas12a platform along with RPA (39°C for 20 min) substantially enhanced analytical sensitivity, enabling reliable detection of as few as 10 copies/µL of viral DNA. The specificity of the assay was rigorously evaluated using individual targets, mixed-template samples, and 90 clinical specimens, demonstrating complete species-specific discrimination without any detectable cross-reactivity. Collectively, the developed single-gene RPA-CRISPR-Cas12a lateral flow assay (LFA) represents a rapid, highly sensitive, and specific point-of-care diagnostic platform, providing visual readout by the naked eye and offering significant potential for decentralized veterinary diagnostics and field-based disease surveillance.},
}

Animals
*Capripoxvirus/isolation & purification/genetics/classification
*CRISPR-Cas Systems
Sensitivity and Specificity
Rapid Diagnostic Tests
*Poxviridae Infections/diagnosis/veterinary/virology
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Systems
Cattle
Goats
Sheep
Goat Diseases/diagnosis/virology
Sheep Diseases/diagnosis/virology

@article {pmid42742174,
year = {2026},
author = {Jerabek, S and Jung, C and Kappy, M and Zhao, Q and Sung, J and Wang, N and Kim, E and Kim, J and Kulmann, MIR and King, MB and McAndrew, MJ and Li, M and Bhatele, S and Isado, M and Jang, HS and Dolezal, M and Prosser, R and Xu, S and Hwang, GH and Pichova, I and Xu, J and Marin, D and Woo, JS and Bae, S and Treff, N and Lapinaite, A and Egli, D},
title = {Highly efficient base editing at PCSK9 and normal human embryo development.},
journal = {Nature},
volume = {658},
number = {8138},
pages = {1289-1300},
pmid = {42742174},
issn = {1476-4687},
mesh = {Humans ; *Gene Editing/methods ; *Proprotein Convertase 9/genetics ; Female ; *Embryonic Development/genetics ; Blastocyst/metabolism/cytology ; CRISPR-Cas Systems/genetics ; DNA Repair/genetics ; DNA Breaks, Double-Stranded ; Alleles ; Chromosome Aberrations ; *Embryo, Mammalian/metabolism/cytology ; Homozygote ; },
abstract = {Cas9-based tools enable programmable DNA lesions for studying repair outcomes, gene function and genome correction. In human embryos, Cas9-induced DNA double-stranded breaks are genotoxic, causing frequent aneuploidy and large deletions[1,2]. Here, we evaluate DNA repair outcomes at nicks and mismatches introduced by base editors at the PCSK9 and HBG loci in human embryos. Delivering ABE8e(V106W) as a protein at fertilization achieved editing at all PCSK9 alleles, supporting development to the blastocyst stage and the derivation of homozygous edited stem cell lines. No insertions or deletions were detected, although rare on-target chromosome breakages and chromosomal abnormalities occurred. Furthermore, editing at bystander and off-target sites was mosaic, and the introduction of the editor as mRNA caused frequent embryo arrest due to guide-independent deaminase activity. Thus, in contrast to Cas9-induced DNA breaks, base-editor-induced lesions are efficiently repaired. However, undesirable consequences for the genome and development can occur, currently precluding the clinical use of base editing in reproduction.},
}

Humans
*Gene Editing/methods
*Proprotein Convertase 9/genetics
Female
*Embryonic Development/genetics
Blastocyst/metabolism/cytology
CRISPR-Cas Systems/genetics
DNA Repair/genetics
DNA Breaks, Double-Stranded
Alleles
Chromosome Aberrations
*Embryo, Mammalian/metabolism/cytology
Homozygote

@article {pmid42744980,
year = {2026},
author = {Jiao, Z and Lin, L and Tang, R and Zhong, CQ and Wu, C and Li, W and Xu, R and Gao, Y and He, P and Wang, C and Zhang, QQ and Liu, S and Yuan, J and Zhang, Q and Li, J},
title = {Histone H2B monoubiquitination drives sexual commitment in malaria parasites.},
journal = {Nature microbiology},
volume = {11},
number = {10},
pages = {2805-2820},
pmid = {42744980},
issn = {2058-5276},
support = {82472314//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82072302//National Natural Science Foundation of China (National Science Foundation of China)/ ; W2411080//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82230077//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Histones/metabolism/genetics ; *Ubiquitination ; Animals ; *Plasmodium falciparum/genetics/metabolism/growth & development ; *Plasmodium yoelii/genetics/metabolism/growth & development/physiology ; Protozoan Proteins/metabolism/genetics ; Humans ; *Malaria/parasitology/transmission ; Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Ubiquitin-Protein Ligases/metabolism/genetics ; Gametogenesis ; Transcription Factor AP-2/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Transmission of malaria parasites to mosquito vectors relies on the successful conversion from asexual blood-stage forms into sexual gametocytes. Protein ubiquitination regulates many cellular processes, including cell differentiation in other eukaryotes, but whether specific ubiquitination machinery mediates Plasmodium sexual conversion is unclear. Here we conduct CRISPR-Cas9 mutagenesis screens to systematically profile Plasmodium yoelii E2 ubiquitin-conjugating enzymes for their impact on parasite development. We demonstrate that an E2 enzyme (Rad6B), and its partner RING-type E3 ligase (Tex1), play determinant roles in gametocytogenesis and transmission to mosquitoes. Deletion of either Rad6B or Tex1 results in severe defects in gametocyte production and oocyst formation. The Rad6B-Tex1 complex catalyses histone H2B monoubiquitination in Plasmodium yoelii and Plasmodium falciparum. H2B monoubiquitination promotes H3K4 tri-methylation occupancy at the promoter region of the gene encoding transcription factor AP2-G, a master regulator of sexual conversion, thereby inducing ap2-g expression and initiating sexual commitment. These findings reveal a key epigenetic mechanism leading to sexual differentiation in both human and rodent malaria parasites, providing potential targets for transmission-blocking interventions.},
}

*Histones/metabolism/genetics
*Ubiquitination
Animals
*Plasmodium falciparum/genetics/metabolism/growth & development
*Plasmodium yoelii/genetics/metabolism/growth & development/physiology
Protozoan Proteins/metabolism/genetics
Humans
*Malaria/parasitology/transmission
Ubiquitin-Conjugating Enzymes/genetics/metabolism
Ubiquitin-Protein Ligases/metabolism/genetics
Gametogenesis
Transcription Factor AP-2/genetics/metabolism
CRISPR-Cas Systems

@article {pmid42754710,
year = {2026},
author = {Maire, A and Ortelli, M and Tkacz, E and Dehio, C and Chassaing, B and Sokol, H and Rolhion, N and Bikard, D},
title = {In vivo CRISPRi screens reveal Escherichia coli functional adaptations in the mouse gut.},
journal = {Nature microbiology},
volume = {11},
number = {10},
pages = {2911-2924},
pmid = {42754710},
issn = {2058-5276},
support = {101044479//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; 51NF40_180541//Fondation pour la Recherche Médicale (Foundation for Medical Research in France)/ ; 51NF40_180541//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {Animals ; *Escherichia coli/genetics/physiology ; Mice ; *Adaptation, Physiological/genetics ; Escherichia coli Infections/microbiology ; Prophages/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Fitness ; Escherichia coli Proteins/genetics ; *CRISPR-Cas Systems ; *Gastrointestinal Tract/microbiology ; },
abstract = {Escherichia coli exhibits remarkable genetic diversity that enables it to adapt to the intestinal environment. Here we establish an in vivo CRISPR interference platform that leverages bacterial gene fitness as a high-resolution functional reporter of E. coli adaptations within mice harbouring a defined minimal microbial community (OligoMM12). The screen revealed that diet profoundly shapes the metabolic landscape of E. coli and the essential gene profile identified cross-feeding interactions. Comparison between a laboratory strain (MG1655), a uropathogenic strain (CFT073) and an adherent-invasive E. coli (AIEC LF82) identified distinct genetic requirements for intestinal colonization, highlighting divergent motility, stress response and respiration strategies. In a host inflammatory environment, we found that AIEC LF82 preferably colonized the small intestine with a mobile genetic element, Gally prophage, playing an important role in modulating fitness. These findings provide a high-resolution genetic atlas of E. coli's functional adaptation and demonstrate the utility of functional genomics to probe the gut environment itself.},
}

Animals
*Escherichia coli/genetics/physiology
Mice
*Adaptation, Physiological/genetics
Escherichia coli Infections/microbiology
Prophages/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Fitness
Escherichia coli Proteins/genetics
*CRISPR-Cas Systems
*Gastrointestinal Tract/microbiology

@article {pmid42814570,
year = {2026},
author = {Cohen Yaron, K and Socol, Y and Yedidi, N and Bejerano-Sagie, M and Ravins, M and Fine-Leebhoff, S and Birimberg-Schwartz, L and Grunewald, M and Hullahalli, K and de Sousa Vieira, A and Waldor, MK and Bartfeld, S and Ben-Yehuda, S and Rosenshine, I},
title = {Dual roles of host N-glycosylation in enteropathogenic Escherichia coli infection.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118043},
pmid = {42814570},
issn = {2211-1247},
mesh = {Humans ; Glycosylation ; *Enteropathogenic Escherichia coli/pathogenicity/physiology/metabolism ; *Escherichia coli Infections/metabolism/microbiology ; *Host-Pathogen Interactions ; Polysaccharides/metabolism ; Escherichia coli Proteins/metabolism ; Bacterial Adhesion ; Host Tropism ; CRISPR-Cas Systems ; },
abstract = {Host factors determine pathogen tropism but knowledge of host genes facilitating enteropathogenic Escherichia coli (EPEC) infection is limited. To identify host determinants required for infection by EPEC, we performed two independent genome-scale CRISPR-Cas9 screens. Both converged on host N-glycosylation as a critical requirement for infection. Genetic or pharmacological inhibition of N-glycan maturation revealed that N-glycosylation promotes infection through two distinct pathways. First, N-linked poly-N-acetyllactosamine glycans serve as receptors for the major EPEC adhesin, the bundle-forming pilus (BFP). Second, perturbation of N-glycosylation compromises host membrane structure, resulting in reduced type III secretion system activity, independently of host cell attachment. Using epithelial monolayers derived from human colon and jejunum organoids, we further show that EPEC attachment inversely correlates with terminal mannose exposure, suggesting glycan composition may be involved in determining intestinal tropism. Together, these findings establish host N-glycan maturation as a key host pathway exploited during enteric infection.},
}

Humans
Glycosylation
*Enteropathogenic Escherichia coli/pathogenicity/physiology/metabolism
*Escherichia coli Infections/metabolism/microbiology
*Host-Pathogen Interactions
Polysaccharides/metabolism
Escherichia coli Proteins/metabolism
Bacterial Adhesion
Host Tropism
CRISPR-Cas Systems

@article {pmid42837260,
year = {2026},
author = {Ma, W and Liu, B and Gao, J and Nie, R and Liu, Z and Wang, W and Wang, Y and Ning, G and Zhao, C and Wang, Y and Yue, X and Ding, D and Ma, X and He, J and Wang, X and Qi, F and Liu, Y and Wang, F and Wang, X and Zhi, D and Wang, D},
title = {Genome-wide CRISPR screening identified hemojuvelin as a non-neuronal receptor for the botulinum neurotoxin serotypes A and C.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118106},
doi = {10.1016/j.celrep.2026.118106},
pmid = {42837260},
issn = {2211-1247},
mesh = {Animals ; *Botulinum Toxins/metabolism ; Mice ; *Botulinum Toxins, Type A/metabolism ; *CRISPR-Cas Systems/genetics ; *GPI-Linked Proteins/metabolism/genetics ; Humans ; Mice, Knockout ; Neuromuscular Junction/metabolism ; Protein Binding ; Sarcolemma/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Botulinum neurotoxins (BoNTs) enter cells primarily through neuronal receptors and gangliosides. However, the mechanism that enables their specific accumulation at neuromuscular junctions (NMJs) remains poorly understood. In this study, a genome-wide CRISPR-Cas9 screen identifies hemojuvelin (HJV), a membrane protein abundant in skeletal muscle, as a non-neuronal receptor for BoNT/A and BoNT/C. Knockout and rescue experiments show that HJV mediates toxin binding by a direct interaction between its C-terminal region (residues 155-393) and the toxin WY loop within the receptor-binding domain (HCR). In mice, HJV knockout markedly reduces toxin enrichment on the sarcolemma and significantly attenuates toxin sensitivity such as digital paralysis and lethality. Thus, HJV captures these toxins on the muscle sarcolemma, establishing a critical prerequisite for efficient recognition and internalization by neuronal receptors at NMJs. These findings further refine the entry mechanism of BoNTs in vivo and offer actionable insights for treating BoNT intoxication and for optimizing their clinical applications.},
}

Animals
*Botulinum Toxins/metabolism
Mice
*Botulinum Toxins, Type A/metabolism
*CRISPR-Cas Systems/genetics
*GPI-Linked Proteins/metabolism/genetics
Humans
Mice, Knockout
Neuromuscular Junction/metabolism
Protein Binding
Sarcolemma/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42849292,
year = {2026},
author = {D'Attilia, C and Bonarrigo, M and Tarkowská, D and Buffagni, V and Hayta, S and Smedley, MA and Masci, S and Palombieri, S and Sestili, F},
title = {CRISPR-Cas9 knockout of IBH1 fine-tunes brassinosteroid metabolism and modulates plant architecture and grain traits in durum wheat.},
journal = {Plant physiology and biochemistry : PPB},
volume = {239},
number = {},
pages = {111831},
doi = {10.1016/j.plaphy.2026.111831},
pmid = {42849292},
issn = {1873-2690},
mesh = {*Triticum/metabolism/genetics/anatomy & histology ; *Brassinosteroids/metabolism ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; *Transcription Factors/genetics/metabolism ; *Edible Grain/genetics/metabolism ; Signal Transduction ; },
abstract = {Brassinosteroids (BRs) are essential steroidal plant hormones that regulate cell elongation, vascular differentiation and reproductive development, thereby shaping plant architecture and grain yield. Fine-tuning BR signalling, rather than altering hormone accumulation, has been proposed as a potential strategy to regulate plant development. Here we investigated the role of IBH1 transcription factor, a negative regulator within the ILI1/PRE1-IBH1 module controlling BR-mediated cell elongation in durum wheat (Triticum turgidum ssp. durum), using a CRISPR-Cas9-mediated knockout approach. IBH1 loss of function was associated with transcriptional changes consistent with enhanced BR signalling, including the coordinated upregulation of key signalling components (BRI1 and BZR1) and downstream regulators (ILI1 and PRE1). Metabolic profiling revealed selective remodelling of BR biosynthesis, characterized by differential accumulation of specific pathway intermediates rather than a general increase in BR content. Phenotypic analyses showed that TdIBH1 mutants exhibited increased plant height, internode elongation, leaf inclination, grain size, and thousand grain weight, while no significant differences in fertility were detected. These findings support a role for TdIBH1 as a conserved negative regulator of BR-mediated growth in durum wheat and suggest that the manipulation of the ILI1/PRE1-IBH1 module may provide a potential strategy to modulate plant architecture and grain-related traits in cereal crops.},
}

*Triticum/metabolism/genetics/anatomy & histology
*Brassinosteroids/metabolism
*Plant Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Gene Knockout Techniques
*Transcription Factors/genetics/metabolism
*Edible Grain/genetics/metabolism
Signal Transduction

@article {pmid42867989,
year = {2026},
author = {Das, M and Banerjee, D and Hossain, A and Jensen, M and Mogre, S and Sun, J and Mao, J and Glick, AB and Salis, HM and Girirajan, S},
title = {Quantifying the impact of genetic mutations on enhancer dynamics.},
journal = {Cell reports},
volume = {45},
number = {10},
pages = {118131},
doi = {10.1016/j.celrep.2026.118131},
pmid = {42867989},
issn = {2211-1247},
mesh = {*Enhancer Elements, Genetic/genetics ; Humans ; *Mutation/genetics ; Gene Expression Regulation ; Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Transcriptional regulation is mediated by enhancers, yet how genetic perturbations alter enhancer activity and gene expression remains unclear. We develop UDI-UMI-STARR-seq, integrating dual indexes and unique molecular identifiers, and combine it with RNA-seq to profile perturbation-associated changes in enhancer activity and gene expression. We apply this approach to 253,632 fragments representing 46,142 cell type-specific candidate enhancers and assess the impact of CRISPR/Cas9-mediated deletion of six transcription factors (ATF2, CTCF, FOXA1, LEF1, TCF7L2, and SCRT1). We identify responsive fragments that are repressed or induced and enriched for p53 family motifs. Enhancer-gene mapping reveals associations between responsive fragments and gene expression changes, including Wnt/p53 targets after ATF2 or LEF1 depletion. A deep learning model recapitulates enhancer grammar, including cooperative motif syntax and flanking sequence context. Applying this framework to 16p12.1 deletion identifies responsive fragments associated with genes involved in neuronal function, providing a scalable readout of enhancer dynamics across genetic perturbations.},
}

*Enhancer Elements, Genetic/genetics
Humans
*Mutation/genetics
Gene Expression Regulation
Transcription Factors/metabolism/genetics
CRISPR-Cas Systems/genetics
Animals

@article {pmid42924445,
year = {2026},
author = {Ono, S and Ono, M and Brettschneider, R and Sauer, D and Müller, K and Balboni, M and van der Heide, M and Schnittger, A},
title = {Reconstructing tissue culture to improve Agrobacterium-mediated transformation of maize.},
journal = {The Plant journal : for cell and molecular biology},
volume = {128},
number = {2},
pages = {},
pmid = {42924445},
issn = {1365-313X},
support = {SCHN 736/20-1//Deutsche Forschungsgemeinschaft/ ; Research Unit 5235//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Zea mays/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Tissue Culture Techniques/methods ; DNA, Bacterial/genetics ; *Agrobacterium/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to insert, delete, or modify genetic information is crucial for mechanistic studies and biotechnological applications. However, efficient genetic transformation remains a major bottleneck for research in maize and many other crops. Here, we report an optimized Agrobacterium-mediated transformation platform based on systematic reconstruction of tissue culture handling in the maize inbred line A188. Refinement of callus induction, selection, and regeneration substantially improved recovery of transgenic plantlets. To distinguish independent T-DNA insertion events, we developed TAFLP (T-DNA amplified fragment length polymorphism), a simple and inexpensive assay that amplifies T-DNA flanking sequences and can be performed using standard laboratory equipment. Our enhanced transformation pipeline was also applicable to the inbred line B104 as well as to hygromycin and G418 selection systems, demonstrating broad utility of our method. We validated the platform for CRISPR/Cas9 mutagenesis and reporter-line generation. Using this approach, we isolated new loss-of-function alleles of the maize-specific MAC1 (MULTIPE ARCHESPORIAL CELLS 1) and F-box protein encoding ACOZ1 (ABNORMAL CHROMOSOME ORGANIZATION IN ZYGOTENE 1), a homolog of the Arabidopsis RMF1/2 (REDUCED MALE FERTILITY 1/2) and rice ZYGO1 (ZYGOTENE 1). The concomitant generation of reporter lines for ACOZ1 and the nuclear lamin-like proteins NCH1 (NMCP/CROWN HOMOLOG1) and NCH2, as well as SUN1 (SUN DOMAIN PROTEIN 1) revealed that the function of this F-box protein class in releasing the somatic constraints of chromosome organization for meiotic recombination is highly conserved in flowering plants.},
}

*Zea mays/genetics
Plants, Genetically Modified/genetics
*Transformation, Genetic
*Tissue Culture Techniques/methods
DNA, Bacterial/genetics
*Agrobacterium/genetics
CRISPR-Cas Systems

@article {pmid42925420,
year = {2026},
author = {Li, Q and Zeng, Z and Zhao, Y and Bi, J and Yan, Q and Wang, C},
title = {TaqW-6BL, a novel gene positively regulates grain WE-AX content in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {11},
pages = {},
pmid = {42925420},
issn = {1432-2242},
support = {32401870//National Natural Science Foundation of China/ ; 251100110200//Henan Province Major Science and Technology Project/ ; },
mesh = {*Triticum/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Genes, Plant ; *Seeds/genetics/chemistry ; Phenotype ; Edible Grain/genetics/chemistry ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {The TaqW-6BL gene, which encodes a TAXI-type inhibitor, influences the water-extractable arabinoxylan content in wheat grains. Water-extractable arabinoxylans (WE-AX) are major dietary fiber components and play a crucial role in wheat end-use quality, especially its nutritional quality. In this study, TaqW-6BL was cloned from parental varieties of a recombinant inbred line (RIL) population, in which a missense mutation (+ 296 bp, T/C) was detected and found to cause a methionine (Met) to threonine (Thr) substitution. Overexpression of TaqW-6BL positively regulated grain WE-AX content, thousand kernel weight (TKW) and mean grain perimeter (MP), with increases of 30.80%, 14.59% and 14.53%, respectively. Conversely, CRISPR/Cas9-mediated knockout of TaqW-6BL led to significant decreases in WE-AX content, TKW and MP. Cytological observations revealed that the aleurone layer of overexpression lines partially proliferated into two layers, whereas knockout lines showed disordered cell arrangement and abnormal morphology. Subcellular localization analysis confirmed that TaqW-6BL protein localizes in the endoplasmic reticulum (ER). The relative expression level and WE-AX content of the favorable haplotype (TaqW-6BL[Avocet]) were significantly higher than those of the unfavorable haplotype (TaqW-6BL[Chilero]) at 14 days and 21 days after flowering (DAF) (P < 0.01). Meanwhile, we developed a functional cleaved amplified polymorphic sequence (CAPS) marker CAPS-6B-296. The wheat varieties with favorable haplotype TaqW-6BL[Avocet] accounted for 68% of 160 wheat varieties. Therefore, our findings provide a valuable gene and a reliable functional marker for nutritional quality breeding in wheat.},
}

*Triticum/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Genes, Plant
*Seeds/genetics/chemistry
Phenotype
Edible Grain/genetics/chemistry
Plants, Genetically Modified
CRISPR-Cas Systems
Mutation, Missense