@article {pmid41152563,
year = {2026},
author = {Erbasan, E and Aliciaslan, M and Erendor, F and Sanlioglu, AD and Sanlioglu, S},
title = {Therapeutic precision gene editing of cholesterol pathways as a gene therapy strategy for cardiovascular disease.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {218-237},
pmid = {41152563},
issn = {1476-5462},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Cardiovascular Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; Proprotein Convertase 9/genetics ; *Hypercholesterolemia/therapy/genetics ; *Cholesterol/metabolism/genetics ; Precision Medicine ; Cholesterol, LDL ; },
abstract = {Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mortality. While established lipid-lowering therapies, primarily statins, are effective for many patients, a significant proportion either fail to achieve optimal LDL-C targets, experience dose-limiting side effects, or face challenges with the long-term adherence required for sustained cardiovascular benefit. The recent emergence and rapid advancement of precise gene editing technologies most notably CRISPR-Cas9 and its advanced variants like base editing and prime editing offer a revolutionary therapeutic paradigm. These tools have the potential to achieve durable modification of the expression or function of genes fundamentally involved in cholesterol metabolism. This comprehensive overview integrates the current knowledge of critical cholesterol regulatory pathways and the main protein targets that are suitable for gene editing. The fundamental mechanisms, relative advantages, and inherent limitations of gene editing platforms and delivery systems for clinical translation are examined. The expanding preclinical data and groundbreaking clinical evidence highlighting the transformative potential of gene editing to achieve significant and lasting reductions in LDL-C, especially through promising therapies like VERVE base editors targeting PCSK9 and ANGPTL3 are critically evaluated. The challenges including off-target effects, delivery efficiency and specificity, long-term safety and durability, complex ethical considerations, and evolving regulatory landscapes that must be rigorously navigated for these therapies to become mainstream clinical practice are thoroughly addressed. Successfully overcoming these challenges could mark the beginning of a new era of personalized, one-time treatments for hypercholesterolemia.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*Cardiovascular Diseases/therapy/genetics
CRISPR-Cas Systems
Animals
Proprotein Convertase 9/genetics
*Hypercholesterolemia/therapy/genetics
*Cholesterol/metabolism/genetics
Precision Medicine
Cholesterol, LDL

@article {pmid41535609,
year = {2026},
author = {Gottimukkala, KSV and Lane, DD and Cunningham, R and Malik, HS and Jwa, Y and Cassidy, ME and Castelli, JMP and Enstrom, MR and Poljakov, K and Gastelum, G and Ho, SH and Tassa, C and Adair, JE},
title = {CRISPR-AuNP: physicochemical optimization of a gold nanoparticle platform for cost-effective and modular non-viral gene editing in HSPCs.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {188-202},
pmid = {41535609},
issn = {1476-5462},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 AI158728/AI/NIAID NIH HHS/United States ; R01 AI167009/AI/NIAID NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {*Gene Editing/methods/economics ; *Gold/chemistry ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Humans ; *Hematopoietic Stem Cells/metabolism ; Cost-Benefit Analysis ; Polyethylene Glycols/chemistry ; },
abstract = {Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.},
}

*Gene Editing/methods/economics
*Gold/chemistry
*CRISPR-Cas Systems
*Metal Nanoparticles/chemistry
Humans
*Hematopoietic Stem Cells/metabolism
Cost-Benefit Analysis
Polyethylene Glycols/chemistry

@article {pmid41637037,
year = {2026},
author = {Hao, J and Gao, X and Light, C and Sun, Y and Lu, S and Tian, Y and Gao, X and Su, Y and Gao, J and Huang, X and Zhang, Q and Wang, J and Hai, R and Hu, W and Wang, G},
title = {Genome-wide CRISPR/Cas9 knockout screen identifies host factors essential for bovine parainfluenza virus type 3 replication.},
journal = {Science China. Life sciences},
volume = {69},
number = {4},
pages = {1301-1316},
pmid = {41637037},
issn = {1869-1889},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems/genetics ; *Virus Replication/genetics ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; *Respirovirus Infections/virology/veterinary/genetics ; Gene Knockout Techniques ; Cell Line ; *Host-Pathogen Interactions/genetics ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of the bovine respiratory disease complex (BRDC), resulting in significant economic losses. Understanding the mechanisms of BPIV3 infection, particularly the entry process, is essential for developing effective control measures. Identifying specific host factors that viruses exploit during their life cycle can reveal critical vulnerabilities for potential antiviral targets. We established a genome-wide CRISPR/Cas9 knockout screen in bovine cells to identify host factors involved in viral infections. Our screen identified several key host factors required for BPIV3 infection, including the sialic acid transporter SLC35A1 and the Sm-like protein LSM12. Further mechanistic analysis revealed that these factors played critical roles at distinct stages of the BPIV3 entry process. These findings not only advance our understanding of how BPIV3 infects host cells but also identify potential host targets for inhibiting infection and developing novel antiviral strategies.},
}

Animals
Cattle
*CRISPR-Cas Systems/genetics
*Virus Replication/genetics
*Parainfluenza Virus 3, Bovine/physiology/genetics
*Respirovirus Infections/virology/veterinary/genetics
Gene Knockout Techniques
Cell Line
*Host-Pathogen Interactions/genetics

@article {pmid41653634,
year = {2026},
author = {Jung, KM and Klein, R and Mony, SI and Chen, PR and Lee, K and Lee, HJ},
title = {Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene.},
journal = {Poultry science},
volume = {105},
number = {4},
pages = {106585},
pmid = {41653634},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Gene Editing/veterinary/methods ; *Genes, Essential ; *CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; Transgenes ; *Avian Proteins/genetics/metabolism ; Cell Line ; Mutagenesis, Insertional ; },
abstract = {Achieving stable and efficient transgene expression is a key challenge in advancing avian genome engineering. Although viral vector-based and piggyBac-mediated transgenesis have been widely used in chickens, both approaches are prone to epigenetic silencing, leading to inconsistent, tissue-specific, and often diminished expression over time. This variability limits used of transgenes requiring robust and long-term expression across multiple tissues. In mammals, site-specific integration into genomic safe harbor loci, such as Rosa26, has enabled stable and predictable transgene expression without disrupting endogenous gene function; however, such strategy has not been established in birds. In this research, we hypothesized that integrating Cas9 into endogenous housekeeping genes (the ACTB and GAPDH) could achieve efficient gene editing in chickens through stable and ubiquitous transgene expression. Using two different approaches, 3'-targeted gene insertion and gene tagging, we inserted Cas9 and GFP cassettes into defined genomic loci in chicken DF-1 cells. Both approaches exhibited stable expression of transgenes in the cells, and functional assays confirmed that Cas9 showed highly efficient nuclease activity following guide RNA delivery. Additionally, we derived single-cell clones stably expressing Cas9, enabling uniform and reproducible genome editing in downstream applications. Targeted insertion of transgenes into active housekeeping genes as candidate safe harbor loci mitigates the limitations of random integration and promoter silencing, offering a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.},
}

Animals
*Chickens/genetics
*Gene Editing/veterinary/methods
*Genes, Essential
*CRISPR-Cas Systems
Animals, Genetically Modified/genetics
Transgenes
*Avian Proteins/genetics/metabolism
Cell Line
Mutagenesis, Insertional

@article {pmid41656178,
year = {2026},
author = {McLamarrah, T and Aral, E and Hoffman, M and Tedstone, J and King, T and Vitko, J and Sebastião, MJ and Escandell, JM and Dias, MM and McCall, I and Machado, D and Cairns, V and DeMaria, C and Scarcelli, JJ},
title = {Evaluation of gene editing in CHO cells using the Cas-CLOVER system.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70108},
doi = {10.1002/btpr.70108},
pmid = {41656178},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Editing/methods ; *Glutamate-Ammonia Ligase/genetics ; *CRISPR-Cas Systems/genetics ; Recombinant Proteins/genetics/biosynthesis ; Cricetinae ; Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; },
abstract = {Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.},
}

CHO Cells
Animals
Cricetulus
*Gene Editing/methods
*Glutamate-Ammonia Ligase/genetics
*CRISPR-Cas Systems/genetics
Recombinant Proteins/genetics/biosynthesis
Cricetinae
Antibodies, Monoclonal/genetics/biosynthesis
Gene Knockout Techniques

@article {pmid41724722,
year = {2026},
author = {Limia, CG and Steffey, V and Cheng, HC and Machado, D and Hart, T and McHargue, MC and Brizzee, C and Crawford, J},
title = {Sequential, chromosome-specific glutamine synthetase double knockout with Cas-CLOVER establishes enhanced CHO platforms for cell line development.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70113},
doi = {10.1002/btpr.70113},
pmid = {41724722},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; *Glutamate-Ammonia Ligase/genetics/metabolism ; Cricetulus ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.},
}

CHO Cells
Animals
*Glutamate-Ammonia Ligase/genetics/metabolism
Cricetulus
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods

@article {pmid41735098,
year = {2026},
author = {Naruse, K and Loosli, F and Ansai, S and Birney, E and Wittbrodt, J},
title = {Medaka: a novel model for analyzing genome-environment interactions.},
journal = {Trends in genetics : TIG},
volume = {42},
number = {4},
pages = {350-361},
doi = {10.1016/j.tig.2025.12.005},
pmid = {41735098},
issn = {0168-9525},
mesh = {Animals ; *Oryzias/genetics ; *Gene-Environment Interaction ; Genome-Wide Association Study ; *Genome ; Quantitative Trait Loci ; CRISPR-Cas Systems ; },
abstract = {Medaka is an established vertebrate model system for biological and biomedical research. It possesses unique features that make it particularly suitable for studying genome-environment interactions. Endemic to habitats spanning from 4 to 40°C and varying salinities, it combines broad ecological adaptability with experimental tractability. Its exceptional tolerance to inbreeding enabled the creation of the Medaka Inbred Kiyosu-Karlsruhe panel-80 near-isogenic, fully sequenced lines derived from a single wild population. More than 100 wild-derived, fully sequenced strains, collected throughout East Asia for more than 40 years, show relatively low intra-strain variation (inbreeding coefficient of >0.75) but high inter-strain variability (SNP rates >4%). Advanced quantification methods facilitate genome-wide association studies and quantitative trait locus mapping. The system's amenability to clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing and emerging epigenomic profiling enables causal validation and regulatory-mechanism discovery. Collectively, medaka offers an unparalleled vertebrate framework for integrating genetics, environment, and epigenetics-bridging evolutionary, biomedical, and population-level perspectives.},
}

Animals
*Oryzias/genetics
*Gene-Environment Interaction
Genome-Wide Association Study
*Genome
Quantitative Trait Loci
CRISPR-Cas Systems

@article {pmid41869962,
year = {2026},
author = {Yang, L and Tan, H and Wang, Y and Zhang, J and Meng, X and Liu, X and Hou, T and Chen, W and Li, F},
title = {Fluidly Confined CRISPR-Magnetic Microbots Empowered Homogeneous Electrochemical Biosensor for Amplified Detection and Discrimination of Cancer-Derived Extracellular Vesicle Subtypes.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10103-10111},
doi = {10.1021/acs.analchem.6c00448},
pmid = {41869962},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Doxorubicin/chemistry ; DNA/chemistry ; *Neoplasms ; Biomarkers, Tumor/analysis ; },
abstract = {Accurate identification and profiling of multiple protein biomarkers on tumor-derived extracellular vesicles (tEVs) are crucial for noninvasive cancer subtyping diagnosis but remain technically challenging due to their high heterogeneity, low abundance in biofluids, and preisolation/purification processes. Herein, we developed a homogeneous electrochemical biosensor empowered by fluidly confined CRISPR-magnetic microbots for the amplified detection and sensitive discrimination of tEV subtypes. The CRISPR-magnetic microbots were constructed by engineering CRISPR/Cas12a and DNA icosahedra/doxorubicin (DNA-ICOS/DOX) on intracellularly gelated magnetic cells (IGMCs). Benefiting from the synergistic effects of spatial confinement and membrane fluidity to elevate the local concentration and collision efficiency, the activity of CRISPR/Cas12a was found to be greatly enhanced on IGMCs. For selective sorting of tEVs, a logic-gated aptamer system was used to orthogonally label tEV subpopulations, which further triggers the trans-cleavage activity of CRISPR/Cas12a, resulting in the release of massive DNA-ICOS/DOX into solution. After magnetic separation, the liberated DOX molecules generate a strong electrochemical signal. Particularly, the CRISPR-magnetic microbots could efficiently reduce the background signal, endowing a significantly improved signal-to-noise ratio. Therefore, by combining the CRISPR-magnetic microbots with the dual-target-guided orthogonal barcoding strategy in a homogeneous electrochemical biosensor, precise identification and sensitive detection of tEVs were successfully achieved. More significantly, this assay achieves accurate cancer subtyping in clinical samples, demonstrating its potential as a robust, noninvasive tool for high-accuracy disease screening, classification, and progression monitoring.},
}

*Biosensing Techniques/methods
Humans
*Extracellular Vesicles/chemistry/metabolism
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Doxorubicin/chemistry
DNA/chemistry
*Neoplasms
Biomarkers, Tumor/analysis

@article {pmid41870471,
year = {2026},
author = {Zhao, X and Wang, Y and Wang, L and Liao, S and Gong, T and Xiong, M and Yu, B and Song, ZL},
title = {Direct RNA Triggering of Cas12a through the Native crRNA Architecture Enables Clinical Nucleic Acids Diagnostics.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9982-9992},
doi = {10.1021/acs.analchem.5c08220},
pmid = {41870471},
issn = {1520-6882},
mesh = {Humans ; Manganese/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; *RNA/metabolism/chemistry ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *MicroRNAs/blood/genetics ; Lung Neoplasms/diagnosis/blood ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for nucleic acid diagnostics, yet its activity is widely considered to be restricted to DNA targets, limiting its applicability for direct RNA detection. Here we report a manganese-ion (Mn[2+])-empowered Cas12a (MEC) platform that overcomes this constraint by allowing the robust RNA-mediated activation of Cas12a. Structural analyses reveal that Mn[2+] strengthens RNA engagement and reorganizes the catalytic center by coordinating RNA phosphates, resulting in an enhancement of trans-cleavage efficiency by 60-fold relative to the Mg[2+] conditions, without compromising sequence specificity. This Mn[2+]-dependent activation mechanism is conserved across multiple Cas12a orthologues (LbCas12a, AsCas12a, FnCas12a), permitting amplification-free detection of RNA with femtomolar sensitivity across diverse targets, particularly the ultrashort abortive transcripts (7 nt). Analysis of clinical serum samples further demonstrates that MEC quantitatively measures circulating miR-21 with performance concordant with reference clinical assays and effectively distinguishes lung cancer patients from healthy individuals. These results reveal an unrecognized role for Mn[2+] in Cas12a biochemistry and establish a simple, versatile, and highly sensitive framework for RNA diagnostics.},
}

Humans
Manganese/chemistry/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry
*RNA/metabolism/chemistry
CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*MicroRNAs/blood/genetics
Lung Neoplasms/diagnosis/blood
*Bacterial Proteins/metabolism/chemistry

@article {pmid41871234,
year = {2026},
author = {Liao, H and Xie, H and Ye, H and Liu, X and Chen, Y and Zhong, R and He, S and Xiao, X and Xie, Z and Shao, Z and Yu, L and Chen, Z},
title = {One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10004-10014},
doi = {10.1021/acs.analchem.5c08249},
pmid = {41871234},
issn = {1520-6882},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Influenza A virus/genetics/isolation & purification ; *Nucleic Acids/analysis ; Staphylococcal Infections/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Isothermal amplification techniques, such as helicase-dependent amplification (HDA) combined with CRISPR, are cutting-edge approaches for nucleic acid detection. In this work, we developed a novel ultrashort mesophilic HDA (termed usHDA) for rapid, highly sensitive nucleic acid amplification at 37 °C and constructed a one-pot usHDA-CRISPR/Cas12 assay. The usHDA is specifically designed for rapid amplification of ultrashort sequences (about 40 nt) at 37 °C within 30 min. This usHDA-CRISPR/Cas12a detection can be completed within 1 h, achieving a limit of detection (LOD) of 5 aM. When tested on 58 clinical specimens from patients infected with respiratory pathogens, this assay identified 41 positive and 17 negative samples for influenza A virus. This assay achieved 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic curve (area under the curve value = 1.00; n = 58) compared with PCR analysis. Furthermore, 24 samples of Staphylococcus infection were detected using usHDA-CRISPR/Cas12a, and the same 100% sensitivity and specificity were achieved. These findings highlighted the strong applicability of our proposed assay for universal nucleic acid detection.},
}

*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Influenza A virus/genetics/isolation & purification
*Nucleic Acids/analysis
Staphylococcal Infections/diagnosis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41873844,
year = {2026},
author = {Wang, Q and Sheng, M and Zheng, Y and Zhang, B and Jin, Z and Zhang, T and Li, Z and Huang, J and Yang, X},
title = {Electrochemiluminescence Biosensing Platform Based on CRISPR/Cas12a and DNA Nanotweezer-Mediated Catalytic Hairpin Assembly Amplification.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9832-9841},
doi = {10.1021/acs.analchem.5c07500},
pmid = {41873844},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Humans ; *DNA/chemistry/genetics ; Catalysis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The detection of microRNAs (miRNAs) biomarkers has great potential in the early diagnosis of acute myocardial infarction (AMI). Herein, we constructed an electrochemiluminescence biosensing platform based on DNA nanotweezer (DNT)-mediated catalytic hairpin assembly (CHA) and CRISPR/Cas12a system for detecting potential AMI biomarker miRNA-133a. DNT, as a programmable molecular scaffold, can precisely organize molecules at the nanoscale and output high signal-to-background ratio detection signals, which is introduced into the construction of sensing platforms. When the target miRNA was presented, the hairpin in DNT was opened, which altered the DNT structure from a closed state to an open state and exposed the catalytic sequence for CHA. Subsequently, a large number of F/A-F duplexes were generated after the addition of fuel strands (F) and antifuel strands (A-F), which served as the target for activating the CRISPR/Cas12a system. The activated Cas12a collaterally cleaved the signal probe (H1) on the electrode surface, causing the labeled Ru(bpy)3[2+] to detach from the electrode surface, resulting in a weakened ECL signal. We found that compared with the general CHA reaction, the DNT-mediated CHA reaction significantly lowers the leakage of the circuit; thus, a high signal-to-background ratio and detection sensitivity can be obtained. Therefore, we developed a highly sensitive biosensing platform for detecting miRNA-133a with a detection limit of 0.12 fM. This sensing strategy provides a new approach for nucleic acid detection and disease diagnosis.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
Humans
*DNA/chemistry/genetics
Catalysis
Nucleic Acid Amplification Techniques
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41881766,
year = {2026},
author = {Li, S and Tang, S and Xu, W and Zhou, J and Li, X and Wang, J},
title = {SIMPLE-CRISPR: A Sample-to-Result Platform for Point-of-Care Detection of Nucleic Acids via a Functionalized Magnetic-Bead-Based CRISPR Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9964-9971},
doi = {10.1021/acs.analchem.5c08178},
pmid = {41881766},
issn = {1520-6882},
mesh = {*Point-of-Care Systems ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Smartphone ; *Point-of-Care Testing ; Magnetic Phenomena ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) systems show great promise for next-generation molecular diagnostics due to their programmability and specificity. Amplification-free CRISPR detection has a strong potential for point-of-care (POC) testing, and the digital format of the assay naturally improves the sensitivity of amplification-free CRISPR detection. However, integrating sample preparation, reaction implementation, and signal readout into a streamlined and user-friendly POC workflow remains a major technical challenge. We address this by developing SIMPLE-CRISPR (Sample-to-Result Integrated platform with Magnetic-bead extraction, Polydisperse droplets, Low-complexity operation, and Engineered smartphone readout), an amplification-free CRISPR-Cas12a system for POC diagnostics. Our innovation features functionalized magnetic-bead extraction for the efficient enrichment of low-abundance nucleic acid targets from large-volume samples with minimal loss, vortex-driven polydisperse emulsification for facile digital droplet generation, and smartphone-integrated fluorescence imaging for on-site portable signal detection. This integrated approach significantly improves sensitivity compared to bulk CRISPR, removes the need for amplification and complex microfluidics, and provides sample-to-result functionality for POC environments. Clinical validation for human papillomavirus type 18 (HPV18) detection confirmed that the assay achieved diagnostic performance consistent with quantitative real-time polymerase chain reaction (qRT-PCR), demonstrating its great potential for accessible POC nucleic acid diagnostics.},
}

*Point-of-Care Systems
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acids/analysis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Smartphone
*Point-of-Care Testing
Magnetic Phenomena

@article {pmid41917051,
year = {2026},
author = {Lu, J and Lai, J and Cheng, L and Zhan, H and Jie, K and Liu, C and Huang, L and Cen, M and Liu, S and Chen, Z and Zhang, Q and Zhang, J and Wu, J and Pan, B and Chen, S and Zhong, J and He, B and Li, H and Chen, X and Lin, T},
title = {Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-71327-w},
pmid = {41917051},
issn = {2041-1723},
abstract = {CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.},
}

@article {pmid41917262,
year = {2026},
author = {Shi, R and Yang, M and Liu, Y and Gao, H and Lin, Z},
title = {Mechanistic basis for selective Csm6-2 activation by cyclic penta-adenylate in a type III CRISPR-Cas system.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41917262},
issn = {1460-2075},
support = {32471255//MOST | National Natural Science Foundation of China (NSFC)/ ; 32271258//MOST | National Natural Science Foundation of China (NSFC)/ ; 2024J02006//| Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation)/ ; },
abstract = {Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the effectors specific to cA5-one of the most abundant cOA species produced during phage infection-have remained unexplored. Here, we report that the CRISPR ribonuclease Csm6 (Csm6-2) from Actinomyces procaprae selectively employs cA5 as its activator. Csm6-2 utilizes its HEPN domain, rather than the CARF domain, to mediate self-limiting cleavage of cOA activators. Cryo-EM structural analyses reveal that Csm6-2 functions as a homotetramer, and disruption of tetramer formation significantly reduces its ribonuclease activity. Although cA6 and cA5 bind Csm6-2 with comparable affinity, only cA5 induces CARF domain closure, stabilizes the tetramer, and remodels the active site in the HEPN domain. In contrast, the sixth AMP of cA6 imposes significant steric hindrance on CARF domain movement, preventing its closure and subsequent allosteric activation. These findings expand our understanding of the cOA signaling diversity and specific cOA recognition mechanisms in type III CRISPR immunity.},
}

@article {pmid41917354,
year = {2026},
author = {Chen, Z and Huang, X and Pi, Y and Jiang, Y},
title = {Method for Generation of adamtsl4 Knock-Out Zebrafish Lines Using CRRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {73-82},
pmid = {41917354},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *ADAMTS Proteins/genetics ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; Phenotype ; Gene Editing/methods ; Mutation ; Humans ; },
abstract = {Congenital ectopia lentis (CEL) is a rare pediatric ocular disorder characterized by zonular fiber defects leading to lens dislocation and is genetically heterogeneous. Among known causes, biallelic mutations in ADAMTSL-4 represent the second most common genetic contributor, frequently associated with ectopia pupillae (EP)-a distinct and clinically significant feature. However, the mechanisms by which ADAMTSL-4 mutations lead to these ocular abnormalities remain poorly understood, partly due to the lack of effective animal models. In this study, we generated adamtsl-4 knock-out zebrafish lines using the CRISPR/Cas9 system. Through microinjection of sgRNA/Cas9 complexes and multigenerational screening, we established stable homozygous mutant lines. These mutants exhibited consistent phenotypes, including lens dislocation into the vitreous body and marked pupillary displacement, faithfully recapitulating human ADAMTSL-4-related EL and EP. This method provides a practical and scalable strategy for generating loss-of-function zebrafish models, with demonstrated utility in recapitulating phenotypes associated with ADAMTSL-4 mutations. Our approach offers a valuable tool for investigating the molecular mechanisms underlying CEL and EP and may support drug screening and therapeutic discovery in the future.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
*ADAMTS Proteins/genetics
Disease Models, Animal
*Zebrafish Proteins/genetics
Phenotype
Gene Editing/methods
Mutation
Humans

@article {pmid41917356,
year = {2026},
author = {Zheng, L and Wu, Z and Zheng, XL},
title = {Generating adamts13[-/-] Zebrafish via CRISPR/Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {93-111},
pmid = {41917356},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *ADAMTS13 Protein/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; },
abstract = {The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*ADAMTS13 Protein/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockout Techniques/methods
Animals, Genetically Modified
Disease Models, Animal
*Zebrafish Proteins/genetics

@article {pmid41918363,
year = {2026},
author = {Yamada, HY and Rao, CV},
title = {Genome-Wide CRISPR Analysis Uncovers Metformin and NSAID Combination as a Chemopreventive Approach in Smoking-Associated Oral Cancer.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {4},
pages = {183-185},
doi = {10.1158/1940-6207.CAPR-26-0004},
pmid = {41918363},
issn = {1940-6215},
mesh = {*Metformin/therapeutic use/pharmacology ; Humans ; *Mouth Neoplasms/prevention & control/etiology/genetics ; *Anti-Inflammatory Agents, Non-Steroidal/therapeutic use/pharmacology ; *Squamous Cell Carcinoma of Head and Neck/prevention & control/genetics/etiology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; CRISPR-Cas Systems ; *Smoking/adverse effects ; Chemoprevention/methods ; Drug Resistance, Neoplasm/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and carries substantial morbidity. Metformin, a widely used antidiabetic agent, shows promise for HNSCC prevention, but resistance arises in a subset of tumors. In a recent issue of Cancer Prevention Research, Hoang and colleagues use CRISPR screening to identify key mediators of metformin resistance, including AMPK and protein kinase A (PKA), and demonstrate that the cyclooxygenase 2-prostaglandin E2 axis acts upstream of PKA. Because this pathway is readily inhibited by common nonsteroidal anti-inflammatory drugs (NSAID), the findings support clinical evaluation of combined metformin and NSAID therapy to improve HNSCC chemoprevention. See related article by Hoang et al., p. 79 .},
}

*Metformin/therapeutic use/pharmacology
Humans
*Mouth Neoplasms/prevention & control/etiology/genetics
*Anti-Inflammatory Agents, Non-Steroidal/therapeutic use/pharmacology
*Squamous Cell Carcinoma of Head and Neck/prevention & control/genetics/etiology
*Antineoplastic Combined Chemotherapy Protocols/therapeutic use
CRISPR-Cas Systems
*Smoking/adverse effects
Chemoprevention/methods
Drug Resistance, Neoplasm/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41919328,
year = {2026},
author = {Singh, P and Sharma, K and Tamrakar, VK and Khare, R and Bhargava, A and Negi, SS},
title = {CRISPR-Cas system: recent advancements in prompt diagnosis of high-risk HPV genotypes in cervical cancer.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {},
doi = {10.1080/14737159.2026.2654503},
pmid = {41919328},
issn = {1744-8352},
abstract = {INTRODUCTION: The CRISPR/Cas system has emerged as a highly versatile platform for diagnosing infectious diseases, particularly viral pathogens. Human papillomavirus (HPV) comprises of more than 200 types, with persistent infection by 14 high-risk genotypes recognized as the primary cause of cervical cancer worldwide. Early and accurate detection of these High-Risk HPV (HR-HPV) types is essential for effective clinical management and prevention of disease progression.

AREAS COVERED: This narrative review was based on literature searches in PubMed, Scopus, and Google Scholar covering studies published between 2015 and 2024. This review summarizes recent advances in CRISPR/Cas based diagnostics for HR-HPV, including both pre-amplification and amplification-free strategies. Integration of CRISPR systems with diverse readout modalities such as colorimetric, fluorescent, electrochemical, and lateral-flow biosensors has enabled rapid, sensitive, and user-friendly detection suitable for point-of-care testing (POCT), particularly in low-resource settings.

EXPERT OPINION: CRISPR/Cas assays demonstrate high sensitivity, specificity, and speed, offering a promising alternative to conventional molecular techniques for HR-HPV detection and genotyping. The convergence of CRISPR diagnostics with artificial intelligence, microfluidics, and affordable biosensors holds significant potential to transform community-level HPV screening. With continued innovation and regulatory support, CRISPR/Cas systems are poised to become indispensable tools for early HR-HPV detection and cervical cancer prevention.},
}

@article {pmid41923586,
year = {2026},
author = {Oh, Y and Lee, H and Jang, S},
title = {Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {64},
number = {3},
pages = {e2512002},
doi = {10.71150/jm.2512002},
pmid = {41923586},
issn = {1976-3794},
support = {//National Research Foundation of Korea/ ; RS-2025-02214910//Ministry of Science and ICT/ ; //Incheon National University/ ; },
mesh = {*CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Bacteriophages/genetics ; *Metabolic Engineering/methods ; *Anti-Bacterial Agents/pharmacology ; *Microbiota/genetics ; *Bacteria/drug effects/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic resistance has become a critical global health challenge due to the decreased efficacy of existing antibiotics and the emergence of multidrug-resistant pathogens. In particular, the rapid horizontal transfer of resistance genes and the diverse mechanisms by which bacteria acquire resistance have significantly undermined the effectiveness of conventional therapeutic strategies, revealing fundamental limitations in current infectious disease management. In this context, synthetic biology provides a promising framework to overcome the limitations of conventional antibiotics by integrating engineering principles with bioengineering approaches, thereby enabling precise and programmable control of biological processes. These synthetic biology-based approaches offer substantial potential for developing sustainable and highly specific antimicrobial strategies. This review comprehensively examines recent advances in synthetic biology-assisted antimicrobial strategies, including CRISPR-Cas systems, bacteriophage engineering, microbiome engineering, and metabolic engineering-driven antibiotic discovery. Collectively, these approaches represent a precision antimicrobial paradigm that enables selective targeting of resistant bacteria while preserving microbiome homeostasis. These strategies also provide new directions for limiting resistance dissemination and guiding the development of next-generation therapeutics.},
}

*CRISPR-Cas Systems
*Synthetic Biology/methods
*Bacteriophages/genetics
*Metabolic Engineering/methods
*Anti-Bacterial Agents/pharmacology
*Microbiota/genetics
*Bacteria/drug effects/genetics
Humans
*Drug Resistance, Bacterial/genetics
*Drug Resistance, Microbial/genetics

@article {pmid40676395,
year = {2026},
author = {Fereydani, NM and Galehdari, H and Hoveizi, E and Alghasi, A and Ajami, M and Andashti, B and Malayeri, A},
title = {Investigating the Efficacy of the CRISPR/Cas9 Gene-Editing System for Targeting the HBB FSC 36-37 (-T) Mutation Locus in Hematopoietic Stem Cells.},
journal = {Molecular biotechnology},
volume = {68},
number = {4},
pages = {1851-1866},
pmid = {40676395},
issn = {1559-0305},
support = {EE/1401.2.24.118699/SCU.ac.ir//Shahid Chamran University of Ahvaz/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *Hematopoietic Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; beta-Thalassemia/genetics/therapy ; Mutation ; *beta-Globins/genetics ; Iran ; },
abstract = {The emergence of genome editing using the CRISPR/Cas9 system has opened up new possibilities and significantly improved the potential for long-term gene therapy of beta-thalassemia. In Iran, FSC 36/37 (-T) is one of the most common mutations among affected individuals, with the highest frequency in the West region (20.8%) and the South-West region (14%). In the context of a proof-of-concept investigation, we present a comprehensive design and assess the efficacy of a CRISPR construct specifically engineered to employ homology-directed repair for targeting the FSC 36/37 (-T) mutation in the HBB gene. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The guide RNAs were transferred to HEK293 cells. T7EI analysis and a non-denaturing PAGE system were employed to assess the effectiveness of the guide RNAs. Hematopoietic stem cells were isolated using the MACS system, and transfection was performed with the Lonza Nucleofector device. The edited cells were monitored using TaqMan-qPCR and RFLP-PCR techniques. Furthermore, the Cas9 enzyme's off-target cleavage sites were meticulously examined to ensure the specificity of the editing process. This research revealed successful genome editing in 30% of the clones analyzed in HEK293 cells and 23.91% of the examined clones in HSCs. Our findings demonstrate the potential of CRISPR/Cas9-mediated genome editing as a promising strategy for addressing genetic mutations associated with thalassemia and other monogenic diseases.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
HEK293 Cells
*Hematopoietic Stem Cells/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
beta-Thalassemia/genetics/therapy
Mutation
*beta-Globins/genetics
Iran

@article {pmid41316685,
year = {2026},
author = {Megarani, DV and Yang, L and Siler, HJ and Quijano Cardé, EM and Martyniuk, CJ and Hick, PM and Becker, JA and Soto, E and Surachetpong, W and Yanong, RPE and Subramaniam, K},
title = {One-Pot RT-LAMP CRISPR/Cas12b Platform for Rapid Detection of Tilapia Lake Virus.},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70087},
doi = {10.1111/jfd.70087},
pmid = {41316685},
issn = {1365-2761},
support = {2023-67015-39481//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; *RNA Virus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/veterinary/methods ; Aquaculture ; *RNA Viruses/isolation & purification ; *Tilapia ; },
abstract = {Tilapia Lake Virus (TiLV) is a significant threat to global tilapia aquaculture, highlighting the need for rapid and accurate diagnostic methods to manage outbreaks and minimise economic losses. This study presents the development and partial validation of a one-pot assay integrating RT-LAMP with the CRISPR/Cas12b system for sensitive and specific TiLV detection. This assay amplifies viral RNA using RT-LAMP, while CRISPR/Cas12b enables a real-time detectable signal. Targeting a conserved region in TiLV segment four, the assay achieves results within 75 min at 62°C, with easy visualisation using a portable fluorescence viewer. It demonstrated high sensitivity, with a 95% limit of detection of 79.6 copies (95% CI: 48-132 copies), and high specificity, with no cross-reaction to other fish RNA or DNA viruses. Based on a validation panel of 261 samples from 9 source populations, the assay exhibited 92% diagnostic sensitivity (95% CI: 87%-96%) and 100% diagnostic specificity (95% CI: 97%-100%). When assessed as a non-lethal sample, gills provided a reliable and less invasive alternative despite lower viral loads compared to internal organs. Therefore, this partially validated one-pot assay is potentially practical for enhancing TiLV detection and disease management in aquaculture systems, especially in field settings and resource-limited laboratories.},
}

Animals
*Fish Diseases/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
*RNA Virus Infections/veterinary/diagnosis/virology
*Molecular Diagnostic Techniques/veterinary/methods
Aquaculture
*RNA Viruses/isolation & purification
*Tilapia

@article {pmid41328622,
year = {2026},
author = {Ma, C and Zhou, Y and Jiang, N and Ren, X and Xu, C and Su, N and Fan, Y and Liu, W},
title = {Development of an RPA-CRISPR-Cas12a Fluorescence Assay for Rapid and Sensitive Detection of Tilapia Parvovirus (TiPV).},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70095},
doi = {10.1111/jfd.70095},
pmid = {41328622},
issn = {1365-2761},
support = {32202994//the National Natural Science Foundation of China/ ; 2023TD46//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2024XT0602//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Tilapia ; *Parvovirus/isolation & purification/genetics ; },
abstract = {Tilapia parvovirus (TiPV) is an emerging pathogen associated with high mortality rates in farmed tilapia, highlighting the urgent need for rapid and accurate diagnostic tools. In this study, we established an RPA-CRISPR/Cas12a detection system targeting the TiPV NS1 gene. The assay conditions were systematically optimised, including 15-min RPA amplification at 39°C, with reagent concentrations of 200 nM Cas12a, 250 nM crRNA and 200 nM ssDNA reporter. Specificity tests showed no cross-reactivity with other tilapia pathogens (TiLV, S. agalactiae) and other aquatic pathogens (LMBRaV, YcCV, GCRV II, WSSV, CyHV-2, SVCV). Sensitivity evaluation revealed a limit of detection (LoD) of 1.97 × 10[1]copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10[3]copies/μL). Clinical validation with 20 tilapia samples demonstrated a 50% positive detection rate for RPA-CRISPR/Cas12a, 15% higher than PCR (35%). This integrated method combines the advantages of RPA and CRISPR-based signal transduction, offering a field-applicable solution for TiPV monitoring in resource-limited aquaculture environments.},
}

Animals
*Fish Diseases/diagnosis/virology
Sensitivity and Specificity
*Parvoviridae Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
*Tilapia
*Parvovirus/isolation & purification/genetics

@article {pmid41636766,
year = {2026},
author = {Abbas, W and Hu, J and Zhu, Y and Xu, L and Mo, B and Liu, L},
title = {Decoding MicroRNA Networks in Plant Vegetative and Reproductive Branching: Mechanisms and Applications for Crop Improvement.},
journal = {Plant, cell & environment},
volume = {49},
number = {5},
pages = {2795-2808},
doi = {10.1111/pce.70429},
pmid = {41636766},
issn = {1365-3040},
support = {32270878//National Natural Science Foundation of China/ ; JCYJ20220531101618038//Shenzhen Basic Research General Project/ ; JCYJ20230808105419038//Shenzhen Basic Research General Project/ ; 2024ZY0132//Central Guidance Funds for Local Science and Technology Development of Inner Mongolia/ ; 868-000003011001//Excellence 2035 Research Program of Shenzhen University/ ; 2023DFT005//Research Team Cultivation Program of Shenzhen University/ ; 2025M784036//China Postdoctoral Science Foundation of China/ ; 2025TQ09A772//Guangdong Special Support Program for Young Talents in Innovation Research of Science and Technology/ ; },
mesh = {*MicroRNAs/genetics/metabolism/physiology ; *Crops, Agricultural/genetics/growth & development ; Gene Expression Regulation, Plant ; Gene Editing ; *RNA, Plant/genetics ; Gene Regulatory Networks ; Reproduction/genetics ; Plant Development/genetics ; CRISPR-Cas Systems ; },
abstract = {Plant branching, encompassing both vegetative and reproductive forms, is a complex and crucial process that shapes overall architecture and determines crop yield and biomass. MicroRNAs (miRNAs) have emerged as master regulators in fine-tuning the intricate genetic and hormonal networks that govern plant branching. This review systematically synthesises recent advances in understanding how miRNA-target gene modules regulate essential pathways to orchestrate the branching patterns. We highlight a central insight that specific miRNA families form hierarchical, stage-specific networks that facilitate the independent optimisation of vegetative and reproductive branching. Furthermore, we explore the potential applications of miRNA manipulation in optimising branching architecture to improve crop yield. By critically evaluating strategies such as artificial miRNAs, target mimics and CRISPR/Cas9 genome editing, we discuss how modulating miRNA networks can engineer ideal plant architecture. Finally, we provide a forward-looking perspective on overcoming challenges in miRNA-based crop improvement, emphasising the integration of single-cell omics and epigenetic insights to achieve precise genetic modifications. This review underscores the transformative potential of miRNAs in designing future crops for enhanced productivity.},
}

*MicroRNAs/genetics/metabolism/physiology
*Crops, Agricultural/genetics/growth & development
Gene Expression Regulation, Plant
Gene Editing
*RNA, Plant/genetics
Gene Regulatory Networks
Reproduction/genetics
Plant Development/genetics
CRISPR-Cas Systems

@article {pmid41914300,
year = {2026},
author = {Wang, W and Li, L and Li, X and Zhang, Q and Liu, Y},
title = {Aptamer- and Ribozyme-Engineered sgRNAs for Conditional Control of CRISPR/Cas9 Function.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {31},
number = {3},
pages = {47300},
doi = {10.31083/FBL47300},
pmid = {41914300},
issn = {2768-6698},
support = {2024B02//Fund for Creative Research of The Second People's Hospital of Foshan/ ; 4SG24185G//Postdoctoral Initial Foundation of Guangdong Medical University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Catalytic/genetics/metabolism ; *Aptamers, Nucleotide/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Synthetic Biology/methods ; Genetic Engineering/methods ; Animals ; },
abstract = {The clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a versatile platform for genome editing, transcriptional regulation, and chromosomal imaging. Recent advances in synthetic biology have enabled the engineering of single guide RNA (sgRNA) to confer conditional responsiveness on the CRISPR/Cas9 system. By integrating functional nucleic acid elements, such as aptamers, ribozymes, and aptazymes, into specific structural regions of the sgRNA, researchers have developed systems that respond to a variety of molecular signals, including small molecules, proteins, and endogenous metabolites. These engineered sgRNAs enable spatiotemporal control of gene editing, activation, repression, and imaging in both prokaryotic and eukaryotic cells. This review summarizes the structural principles, design strategies, and applications of condition-responsive CRISPR/Cas9 systems, highlighting their potential in synthetic biology, disease modeling, and therapeutic development. Current challenges and future directions for improving the specificity, efficiency, and applicability of these systems are also discussed.},
}

*CRISPR-Cas Systems/genetics
*RNA, Catalytic/genetics/metabolism
*Aptamers, Nucleotide/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Synthetic Biology/methods
Genetic Engineering/methods
Animals

@article {pmid41914499,
year = {2026},
author = {Davidge, B and Carnes, J and Lewis, I and Rodshagen, T and Tracy, M and McDermott, SM and Stuart, KD},
title = {KREPA6 functions in RNA editing catalytic complex structural organization and gRNA utilization in Trypanosoma brucei.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41914499},
issn = {1362-4962},
support = {R01 AI014102/AI/NIAID NIH HHS/United States ; AI014102/GF/NIH HHS/United States ; //Seattle Children's Research Institute/ ; 2140153//NSF/ ; },
mesh = {*Trypanosoma brucei brucei/genetics/metabolism/growth & development ; *RNA Editing ; *Protozoan Proteins/genetics/metabolism/chemistry ; *RNA, Guide, Kinetoplastida/metabolism/genetics ; Amino Acid Substitution ; RNA, Protozoan/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {Functional mitochondrial mRNAs in Trypanosoma brucei are generated by the post-transcriptional guide RNA (gRNA) directed insertion and deletion of uridine residues, called RNA editing, that is catalyzed by three closely related multiprotein RNA Editing Catalytic Complexes (RECCs). These RECCs contain a common set of 12 proteins including KREPA6 which is largely comprised of an oligonucleotide binding (OB)-fold domain with a predicted intrinsically disordered region (IDR) at its C-terminus. Here we show that certain single amino acid substitutions throughout KREPA6 or deletion of the IDR inhibit the growth and viability of bloodstream form (BF) parasites. These mutations variously impact RECC structure, many alter but do not eliminate RNA editing, and some result in differential utilization of gRNAs. The results indicate that KREPA6 protein has multiple functions some of which stem from its interactions with multiple RECC proteins and perhaps with substrate RNA in each of the three different RECCs. These functions likely involve dynamic interactions of KREPA6 with key domains of other RECC proteins, other editing proteins, and with messenger RNA/gRNA substrates during the multiple catalytic and noncatalytic steps that occur during the complicated editing process.},
}

*Trypanosoma brucei brucei/genetics/metabolism/growth & development
*RNA Editing
*Protozoan Proteins/genetics/metabolism/chemistry
*RNA, Guide, Kinetoplastida/metabolism/genetics
Amino Acid Substitution
RNA, Protozoan/metabolism
*RNA, Guide, CRISPR-Cas Systems/metabolism

@article {pmid41914500,
year = {2026},
author = {Liu, H and Jiao, K and Hao, A and Li, C and Hou, J and Lu, X and Liu, M and Qi, Q},
title = {Enhancement of single-stranded template annealing activity by Rad52 during repair of CRISPR-induced dsDNA breaks.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41914500},
issn = {1362-4962},
support = {U23A20268//National Natural Science Foundation of China/ ; ZR2025MS440//Shandong Provincial Natural Science Foundation/ ; SKLMTFCP-2023-03//SKLMT Frontiers and Challenges Project/ ; },
mesh = {*Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *DNA, Single-Stranded/genetics/metabolism ; *DNA End-Joining Repair ; Gene Editing/methods ; Yarrowia/genetics ; *CRISPR-Cas Systems ; DNA Repair ; },
abstract = {Single-strand annealing protein (SSAP)-mediated recombination engineering has become a powerful tool for bacterial genome editing. However, in most eukaryotes, its efficiency is constrained by the dominant non-homologous end joining (NHEJ) repair pathway and the limited activity of exogenous SSAPs. Here, in the typically NHEJ-dominated yeast Yarrowia lipolytica, we found that 18.7% of Cas9-induced double-strand breaks (DSBs) were precisely repaired upon provision of single-stranded oligonucleotide templates, even in the absence of recombinase overexpression, indicating the presence of an endogenous eukaryotic SSAP-mediated recombination activity. Overexpression of recombination-related proteins revealed that Rad52 plays a key role in single-strand annealing. Structural truncation of Rad52(1-300) boosted genome-editing efficiency to 96.3%, comparable to that achieved by disrupting NHEJ via Ku70 deletion. Our ESTAR platform (enhancement of single-stranded template annealing activity by Rad52) enables precise and efficient genome editing, including small-fragment insertions, deletions, and replacements, as well as large-fragment deletions exceeding 20 kb. This gene-editing technology was further validated in Saccharomyces cerevisiae and other non-conventional yeast, offering new insights into the single-stranded DNA annealing step during the repair of Cas9-induced DSBs.},
}

*Rad52 DNA Repair and Recombination Protein/genetics/metabolism
*DNA Breaks, Double-Stranded
*DNA, Single-Stranded/genetics/metabolism
*DNA End-Joining Repair
Gene Editing/methods
Yarrowia/genetics
*CRISPR-Cas Systems
DNA Repair

@article {pmid41914596,
year = {2026},
author = {Ma, B and Wang, X and Cai, X and He, M and Lai, S and Zhang, P and Xu, J},
title = {Isothermal amplification and CRISPR/Cas one-pot detection systems: strategies and prospects.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01976a},
pmid = {41914596},
issn = {1759-9679},
abstract = {Molecular diagnostic techniques, known for their high sensitivity and specificity, are now the gold standard for detecting pathogen nucleic acids and are crucial in precision medicine. Traditional methodologies, including polymerase chain reaction (PCR) and gene sequencing, offer exceptional analytical precision and reliability. Nevertheless, their applicability for point-of-care testing (POCT) is limited due to the high cost of equipment, prolonged detection durations, and reliance on controlled laboratory settings. The CRISPR/Cas system is considered as the next-generation of nucleic acid-based molecular diagnostic techniques, attributed to its highly specific recognition of target nucleic acids, programmability, high sensitivity, and suitability for POCT. The one-pot CRISPR assay integrates the pre-amplification of DNA or RNA with CRISPR/Cas detection into a single reaction system. This consolidation streamlines experimental workflows, enhances the practicality of POCT, and minimizes the risk of aerosol contamination. Consequently, this method has received tremendous attention from researchers. However, challenges remain due to incompatibilities between isothermal amplification and the CRISPR/Cas system, including mutual interference, temperature mismatches, and buffer incompatibility. This paper reviews recent advancements in one-pot CRISPR/Cas detection strategies, highlighting potential solutions such as physical isolation, microfluidic integration, optimization of the reaction systems, modification of crRNA and PAM sequences and engineering of nucleases. Additionally, it explores the potential applications and challenges associated with these systems in the development of high-performance POCT platforms.},
}

@article {pmid41915142,
year = {2026},
author = {Gottipamula, S and Seetharam, RN and Nk, V},
title = {"Primed for Repair: Harnessing Hypoxia, Mechanobiology, and Gene Editing to Enhance MSC Potency and Clinical Translation".},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {41915142},
issn = {2629-3277},
}

@article {pmid41915857,
year = {2026},
author = {Ahmad, Z and Ramakrishnan, M and Varshney, RK and Shahzad, A and Rehman, S and Pant, B and Wei, Q},
title = {WUSCHEL Transcription Factor: From Stem Cell Maintenance to Crop Improvement.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e19705},
doi = {10.1002/advs.202519705},
pmid = {41915857},
issn = {2198-3844},
support = {32471977//National Natural Science Foundation of China/ ; 32071848//National Natural Science Foundation of China/ ; BK20231289//Natural Science Foundation of Jiangsu Province/ ; Y20240114//Young Foreign Talent Program/ ; QN2022014012L//Young Foreign Talent Program/ ; 163100036//Metasequoia Faculty Research Start-Up Funding/ ; 163100028//Metasequoia Faculty Research Start-Up Funding/ ; JC2019004//Natural Science Foundation For Distinguished Young Scholars of Nanjing Forestry University/ ; 202211//Project for Ground breaking Achievements of Nanjing Forestry University/ ; },
abstract = {The WUSCHEL (WUS) transcription factor, long recognized as a master regulator of stem cell maintenance in the shoot apical meristem (SAM), has expanded in significance as a multifaceted tool in plant biotechnology. With an emphasis on its new uses in crop regeneration, somatic embryogenesis (SE), stress tolerance, and developmental regulation in cereals, legumes, and other plant species, this review summarizes recent developments on WUS function outside of Arabidopsis. We emphasize how insights from WUS biology can be translated into practical strategies to improve yield, adaptability, and resilience, while also enhancing in vitro tissue culture systems. The objective of this review is to establish WUS as a crucial molecular target for future crop genetic improvement and sustainable farming methods by highlighting the current knowledge gaps and suggesting future directions.},
}

@article {pmid41917016,
year = {2026},
author = {Gong, X and Pożoga, M and Boyer, JB and Xue, Y and Meinnel, T and Bange, T and Giglione, C and Hell, R and Wirtz, M},
title = {The ribosome-associated N-terminal acetyltransferase B coordinates global proteostasis and autophagy in plants by creating Ac/N-degrons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41917016},
issn = {2041-1723},
support = {496871662//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 544882710//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 5041140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ANR-17-CAPS-0001-01//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-20-CE92-0040//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Autophagy/genetics ; *Proteostasis ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; Acetylation ; *Ribosomes/metabolism ; *N-Terminal Acetyltransferase B/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; Ubiquitin/metabolism ; Proteomics ; Proteome/metabolism ; Mutation ; CRISPR-Cas Systems ; Degrons ; },
abstract = {The N-terminal acetyltransferase B (NatB) acetylates ~20% of the eukaryotic proteome. However, the role of NatB-mediated N-terminal acetylation (NTA) for the regulation of the proteome fate remains unclear in eukaryotes. In this study, we demonstrate that CRISPR-Cas9-mediated deletion of NatB activity in plants results in significantly lowered global protein turnover due to decreased ubiquitin-proteasome system (UPS) activity and protein translation. Quantitative proteomics uncovers that NatB substrates are significantly enriched in the fraction of stabilized proteins in natb mutants. We provide direct evidence that the absent NTA of KIN11, a subunit of the autophagy-controlling energy sensor SnRK1, protects it from UPS-mediated destruction. The resulting accumulation of KIN11 is responsible for the increased resistance of natb mutants to energy limitation induced by prolonged darkness. Our findings establish NatB as a central regulator of UPS-autophagy interplay and highlight its role in maintaining proteome stability and enabling dynamic stress responses in plants.},
}

*Autophagy/genetics
*Proteostasis
*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/metabolism/genetics
Acetylation
*Ribosomes/metabolism
*N-Terminal Acetyltransferase B/metabolism/genetics
Proteasome Endopeptidase Complex/metabolism
Ubiquitin/metabolism
Proteomics
Proteome/metabolism
Mutation
CRISPR-Cas Systems
Degrons

@article {pmid41192497,
year = {2026},
author = {Wang, X and Chen, Y and Hu, Y and Luo, S and Wang, S and Mao, B and Peng, C and Chen, C and Pan, W and Yan, H and Liao, J and Zhou, Q and Li, W},
title = {Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {4},
pages = {732-743},
doi = {10.1016/j.jgg.2025.10.007},
pmid = {41192497},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA Probes/genetics/chemistry ; Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; Influenza A virus/genetics/isolation & purification ; Papillomavirus Infections/diagnosis/virology ; },
abstract = {CRISPR-based nucleic acid detection technologies have revolutionized infectious disease detection and environmental monitoring by leveraging RNA-DNA complementarity to enable rapid, precise, and cost-effective detection of targets. However, achieving multitarget detection in one tube still presents challenges that necessitate further research. Here, we develop a nucleic acid detection module based on the CRISPR-Cas12i system. Importantly, we find that Cas12i and AapCas12b exhibit opposite trans-cleavage preferences for asymmetrically phosphorothioate-modified single-strand DNA probes, enabling the development of an effective dual-target nucleic acid detection platform by combining these two Cas12 nucleases in one tube. Moreover, this dual-target detection platform exhibits high specificity and sensitivity in genotyping the nucleic acid targets of human papillomavirus (HPV) 16 and HPV18, as well as Influenza A virus (FluA) and Respiratory syncytial virus. Notably, combined with loop-mediated isothermal amplification, this platform achieves high detection rates for clinical samples (18/18 FluA and 18/18 GAPDH internal reference detection rate). Taken together, these results can broaden the application of CRISPR-based Cas12 proteins for multi-target nucleic acid detection in one tube.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA Probes/genetics/chemistry
Nucleic Acid Amplification Techniques/methods
Human papillomavirus 16/genetics/isolation & purification
Human papillomavirus 18/genetics/isolation & purification
Influenza A virus/genetics/isolation & purification
Papillomavirus Infections/diagnosis/virology

@article {pmid41260247,
year = {2026},
author = {Han, F and Lu, Y and Zhang, Y and Ma, X and Tong, C and Wei, J and Guo, Y and Liu, C and Wu, Z and Han, W and Wang, Y},
title = {Intrinsic NPRL2 and NPRL3 regulate the sensitivity of B-cell malignancies to CAR-T cell therapy.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {4},
pages = {655-666},
doi = {10.1016/j.jgg.2025.11.007},
pmid = {41260247},
issn = {1673-8527},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; Animals ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology ; Cell Line, Tumor ; *Lymphoma, B-Cell/therapy/genetics/immunology/pathology ; CRISPR-Cas Systems ; Mechanistic Target of Rapamycin Complex 1/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Although chimeric antigen receptor (CAR) T-cell therapy has markedly improved outcomes for many patients with B-cell malignancies, a subset experiences limited benefit due to primary or secondary resistance. Building on CRISPR/Cas9 genome-wide screening in malignant B-cells, we identify NPRL2 and NPRL3 as key regulators of tumor sensitivity to CAR-T cytotoxicity. This study aims to investigate the impact and mechanisms of tumor-intrinsic NPRL2 and NPRL3 on the efficacy of CAR-T cell therapy. In a tandem CD19/20 CAR-T clinical trial for relapsed/refractory (R/R) B-cell lymphoma (NCT03097770), high tumor NPRL2 or NPRL3 expression correlates with therapeutic resistance in patients. Consistently, in vitro experiments confirm that tumor cells overexpressing NPRL2/NPRL3 exhibit resistance to CAR-T-mediated cytolysis. Mechanistically, NPRL2/NPRL3 suppresses mTORC1 activity within tumor cells, negatively regulating the conjugation between tumor cells and CAR-T cells, consequently impairing CAR-T cell activation and cytotoxic function, ultimately facilitating immune escape. As therapeutic strategies, either genetic ablation of tumor-intrinsic NPRL2/NPRL3 or pharmacological activation of mTORC1 enhances CAR-T cell activation, cytotoxic degranulation, and tumor clearance both in vitro and in vivo. In conclusion, targeting tumor NPRL2/NPRL3 or directly activating mTOR represents a promising combinational strategy to potentiate CAR-T efficacy and overcome resistance in clinical practice.},
}

Humans
*Immunotherapy, Adoptive/methods
Animals
Mice
*Receptors, Chimeric Antigen/genetics/immunology
Cell Line, Tumor
*Lymphoma, B-Cell/therapy/genetics/immunology/pathology
CRISPR-Cas Systems
Mechanistic Target of Rapamycin Complex 1/metabolism
Xenograft Model Antitumor Assays

@article {pmid41642668,
year = {2026},
author = {Takenaka, R and Albanese, EH and Simmerman, SM and Verghese, S and Maddox, MAE and de la Cruz, AF and Young, JM and Schmidt, CA and Rieder, LE and Malik, HS},
title = {The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.},
journal = {Genetics},
volume = {232},
number = {4},
pages = {},
doi = {10.1093/genetics/iyag036},
pmid = {41642668},
issn = {1943-2631},
support = {T32 GM007270/GM/NIGMS NIH HHS/United States ; //NIH IRACDA/ ; F32GM140778//NIH NRSA/ ; R00HD092625//NIH NRSA/ ; R35GM142724//NIH NRSA/ ; R01GM074108//NIH NRSA/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *Histones/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics ; Female ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Oocytes/metabolism ; Maternal Inheritance ; Male ; },
abstract = {The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created 2 new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that loss of ao causes maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. Our data indicate that ao genetically interacts with the heterochromatin, as previously suggested. However, contrary to a prior study, we detected neither Ao localization to histone genes nor ao repression of core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.},
}

Animals
*Histones/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
*Drosophila melanogaster/genetics
Female
Heterochromatin/genetics/metabolism
CRISPR-Cas Systems
*Oocytes/metabolism
Maternal Inheritance
Male

@article {pmid41911152,
year = {2026},
author = {Wu, Y and Qi, Y and Chen, Y and Liu, D and Liu, Q and Shang, X},
title = {CrisprPr: a hybrid-driven framework for CRISPR/Cas9 off-target prediction with analysis of prior-information updates.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {2},
pages = {},
pmid = {41911152},
issn = {1477-4054},
support = {62433016//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Deep Learning ; Computational Biology/methods ; Algorithms ; },
abstract = {CRISPR/Cas9 specificity is critically affected by off-target effects. However, the complex patterns of mismatches and their combinations at off-target sites remain difficult to capture, and existing approaches show limited capacity to identify informative features. Here, we present CrisprPr, a hybrid-driven off-target prediction framework that integrates both prior information and data-driven modeling to improve the characterization of off-target activity. CrisprPr employs a synchronous updating strategy that jointly optimizes prior-knowledge and deep-learning modules, together with multi-source integration, to deliver accurate and stable off-target predictions. Evaluations on independent test sets indicate that CrisprPr achieves competitive predictive performance and generalization compared with existing deep learning methods, with statistically significant improvements observed on several datasets. Beyond predictive performance, its analysis module examines the patterns of prior embedding-space updates to reveal distinctive target-site features supported by literature evidence. Overall, CrisprPr proposes a novel framework that demonstrates competitive predictive performance while offering new insights into the characteristics of off-target effects.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
Deep Learning
Computational Biology/methods
Algorithms

@article {pmid41911457,
year = {2026},
author = {Waild, NC and Ciesla, JH and Schafer, XL and Munger, J},
title = {Cellular antibody affinity-based CRISPR screening identifies JUNB as a broadly acting antiviral factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2534154123},
doi = {10.1073/pnas.2534154123},
pmid = {41911457},
issn = {1091-6490},
support = {AI150698//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI118689//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI184380/AI/NIAID NIH HHS/United States ; GM068411//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 AI181865/AI/NIAID NIH HHS/United States ; AI181865//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Cytomegalovirus/genetics/immunology ; *Cytomegalovirus Infections/genetics/immunology/virology ; *CRISPR-Cas Systems ; *Antibody Affinity ; *Transcription Factors/genetics/metabolism/immunology ; Viral Proteins/genetics/metabolism ; Virus Replication ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; },
abstract = {CRISPR screening is a powerful approach to identify genetic perturbations that impact viral infection. However, most virus-focused CRISPR screens utilize selection strategies that limit the ability to identify genes important for infection. Here, we developed a CRISPR screening pipeline to identify cellular determinants of human cytomegalovirus (HCMV) infection based on virally induced remodeling of cellular antibody affinity (VIRCAA), which is scalable for large libraries and can identify cellular genes that impact HCMV infection at different life cycle stages. We utilized this pipeline to interrogate proteomic and transcriptomic datasets associated with the HCMV UL26 protein, which blocks antiviral signaling during infection. We find that JUNB drives antiviral gene expression, induces protein ISGylation, and suppresses diverse viral infections. Further, UL26 interacts with JUNB and suppresses JUNB-mediated condensation of viral DNA replication compartments. These results highlight the VIRCAA pipeline's utility for identifying important determinants of viral infection.},
}

Humans
*Cytomegalovirus/genetics/immunology
*Cytomegalovirus Infections/genetics/immunology/virology
*CRISPR-Cas Systems
*Antibody Affinity
*Transcription Factors/genetics/metabolism/immunology
Viral Proteins/genetics/metabolism
Virus Replication
*Clustered Regularly Interspaced Short Palindromic Repeats
HEK293 Cells

@article {pmid41911458,
year = {2026},
author = {Richards, L and Lee, D and Wiktor, J and Truedson, A and Cederblad, J and Jones, D},
title = {Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2525424123},
doi = {10.1073/pnas.2525424123},
pmid = {41911458},
issn = {1091-6490},
support = {2020-05137//Swedish Research Council/ ; CTS 21:1334//Carl Tryggers Foundation/ ; },
mesh = {*Plasmids/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Kinetics ; *Escherichia coli/genetics ; Conjugation, Genetic ; Single-Cell Analysis ; },
abstract = {Understanding and manipulating the spread of mobile genetic elements represents a great challenge with potential benefits across synthetic biology, agriculture, and medicine. A key part of this challenge is the multiple scales in play, from the molecular kinetics of defense systems such as CRISPR-Cas, to single-cell variability in immunity levels, to spatial structure in bacterial populations. In this work, we use a time-lapse, imaging-based approach to characterize conjugative plasmid dynamics at the molecular, single-cell, and population levels. By fluorescently tagging the conjugative plasmid RP4 and CRISPR-Cascade complexes, we quantify population dynamics as a function of spacer target number, Cascade expression level, and the presence of plasmid addiction modules. Using single-cell tracking, we report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and quantify the effect of Cascade expression variability on plasmid clearance kinetics. Finally, using a spatially resolved, agent-based model, we show that plasmid population dynamics can be successfully predicted using these single-cell biophysical parameters as inputs. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity, and plasmid cost-benefit tradeoffs. The imaging and analysis techniques used here will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.},
}

*Plasmids/genetics/metabolism
*CRISPR-Cas Systems/genetics
Kinetics
*Escherichia coli/genetics
Conjugation, Genetic
Single-Cell Analysis

@article {pmid41912937,
year = {2026},
author = {Zhang, D and Wang, H},
title = {Liquid Biopsy in Uterine Leiomyosarcoma: Current Biomarkers, Emerging Technologies, and Future Perspectives.},
journal = {Current oncology reports},
volume = {28},
number = {1},
pages = {},
pmid = {41912937},
issn = {1534-6269},
mesh = {Humans ; *Leiomyosarcoma/diagnosis/genetics/pathology/blood ; Liquid Biopsy/methods ; *Uterine Neoplasms/diagnosis/genetics/pathology/blood ; Female ; *Biomarkers, Tumor/genetics/blood ; Neoplastic Cells, Circulating/pathology ; Circulating Tumor DNA/blood ; },
abstract = {PURPOSE OF REVIEW: Uterine leiomyosarcoma (uLMS) is a rare but aggressive malignant mesenchymal tumor, accounting for 2-5% of uterine malignancies. Because its symptoms and imaging features often resemble those of benign uterine leiomyoma (LM), accurate preoperative diagnosis remain difficult. This review summarizes recent advances in liquid biopsy for uLMS and explores its potential for early detection, molecular characterization, and treatment monitoring.

RECENT FINDINGS: Liquid biopsy enables minimally invasive detection of tumor-derived components such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), non-coding RNAs, and extracellular vesicles (EVs). Recurrent mutations in TP53, RB1, and ATRX have been identified through ctDNA analysis, while CTCs, ncRNAs, and EVs provide complementary information for monitoring tumor dynamics and therapeutic response. Emerging technologies including CRISPR-Cas systems, nanotechnology, electrochemical biosensors, and multi-omics integration enhance detection sensitivity and specificity. Liquid biopsy holds promise for improving uLMS diagnosis and management. However, standardization and biomarker validation remain essential to achieve reliable clinical translation and enable earlier, more precise treatment strategies.},
}

Humans
*Leiomyosarcoma/diagnosis/genetics/pathology/blood
Liquid Biopsy/methods
*Uterine Neoplasms/diagnosis/genetics/pathology/blood
Female
*Biomarkers, Tumor/genetics/blood
Neoplastic Cells, Circulating/pathology
Circulating Tumor DNA/blood

@article {pmid41850995,
year = {2026},
author = {Liu, Y and Niu, X and Wei, W},
title = {Decoding disease-relevant variants with base and prime editors at scale.},
journal = {Trends in biochemical sciences},
volume = {51},
number = {4},
pages = {392-409},
doi = {10.1016/j.tibs.2026.02.001},
pmid = {41850995},
issn = {0968-0004},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Variation ; CRISPR-Cas Systems ; *Genomics/methods ; Genome, Human ; },
abstract = {Interpreting variants of uncertain significance remains a central challenge in human genomics. Base and prime editors have launched a new era of precision functional genomics, enabling programmable, double-strand break-free introduction of point mutations and small indels directly within the genome. Here, we review the technological evolution of these editors and their transformative application in high-throughput functional screens. We highlight how base and prime editing platforms systematically annotate clinical variants, reveal mechanisms of drug resistance and immune evasion, and dissect fundamental biological processes at single-nucleotide resolution. Crucially, we address current challenges and future perspectives for precision editing screens. By enabling causal genotype-to-phenotype mapping, precision editing screens are redefining genomic variation interpretation and accelerating its translation into precision diagnostics and therapeutics.},
}

Humans
*Gene Editing/methods
*Genetic Variation
CRISPR-Cas Systems
*Genomics/methods
Genome, Human

@article {pmid41909947,
year = {2026},
author = {Hille, F and Wang, C and Finstermeier, K and Beerens, D and Schmidt, K and Skibbe, M and Charpentier, E},
title = {Insights into spacer acquisition of the type V-A CRISPR-Cas system of Francisella novicida U112.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909947},
issn = {1362-4962},
support = {//Alexander von Humboldt Foundation/ ; //German Research Foundation/ ; //Max Planck Society/ ; },
mesh = {*Francisella/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; DNA, Single-Stranded/metabolism/genetics ; Plasmids/genetics ; Bacterial Proteins/metabolism/genetics ; Integrases/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Prophages/genetics ; },
abstract = {CRISPR-Cas systems immunize prokaryotic cells through a CRISPR adaptation process, in which short DNA fragments from foreign elements are acquired and integrated into the CRISPR array. Here, we investigated the spacer acquisition mechanism of the type V-A CRISPR-Cas system from Francisella novicida U112. We characterized the Cas1-Cas2 integrase in vitro and elucidated the sequence requirements of the pre-spacer and the CRISPR array for optimal spacer incorporation. We demonstrated that Cas2 coordinates metals at its active site to facilitate full-site spacer integration. Furthermore, we introduced this spacer acquisition system into Escherichia coli cells and observed that, in vivo, all Cas proteins are required for efficient type V-A adaptation, with Cas12 significantly improving adaptation efficiency. We showed that spacers were acquired preferentially from plasmids encoding cas genes, and from genomic regions of prophages and origins of replication. In addition, we found that Cas4 possesses a 3'-5' exonuclease activity against single-stranded DNA and an ATP-independent unwinding activity towards double-stranded DNA. Cas4 interacts with the Cas1-Cas2 complex and processes pre-spacers in a PAM-dependent manner. The presence of Cas4 in vivo ensures that new spacers are derived from DNA immediately downstream of a 5'-TTTN-3' PAM, which is critical for targeting invaders.},
}

*Francisella/genetics/enzymology
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Escherichia coli/genetics
DNA, Single-Stranded/metabolism/genetics
Plasmids/genetics
Bacterial Proteins/metabolism/genetics
Integrases/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Prophages/genetics

@article {pmid41909950,
year = {2026},
author = {Deng, J and Zhou, J and Xiang, H and Li, X and Han, X and Weng, Z and Jia, J and Shao, Y and Sima, Y and Niu, M and Li, D and Ouyang, H and Xu, B and Pang, D and Yang, L and Yuan, H},
title = {Regulated transformation system (RTS): sddi-mediated programmable shut-off and mode switching of base editors.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909950},
issn = {1362-4962},
support = {20260102218JC//Jilin Provincial Science and Technology Development Plan Project/ ; 2025YFF1000800//National Key Research and Development Program of China/ ; 32202754//National Natural Science Foundation of China/ ; 32372962//National Natural Science Foundation of China/ ; 20230205117RC//Jilin Province science and technology development plan project/ ; },
mesh = {Humans ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics/metabolism ; Cytosine/metabolism ; HEK293 Cells ; Adenine/metabolism ; Cell Line ; DNA, Single-Stranded/metabolism/genetics ; },
abstract = {Orthogonal and externally controllable base editors are critical for safe multiplexed single-nucleotide manipulation in vivo. Here, we identify ~140-aa miniature deaminase inhibitors (Sddis) that bind cognate single-stranded DNA deaminases (Sdds) with high affinity and specificity, occluding their DNA-binding surfaces to completely inhibit C-to-T activity. Based on these inhibitors, we engineer an adenine and cytosine base editing-regulated transformation system (ACBE-RTS). This platform features two inactive dSdds fused to nCas9 as docking arms, with effector modules provided by doxycycline-inducible SviSddi-SflSdd (CBE) and cumate-inducible Air1Sddi-ABE8e (ABE) fusions. Small-molecule regulation enables switching among four modes (OFF, CBE, ABE, ACBE), achieving up to 43.4% C-to-T or 42.9% A-to-G editing at four endogenous human sites. Using a 4000-member sgRNA library in MARC-145 cells stably expressing ACBE-RTS, a three-round screening identified four key amino acids in monkey CD163 that reduced replication of highly pathogenic PRRSV by >100-fold and eliminated detectable viral-antigen staining. Compact and multi-mode switchable on a single Cas9 scaffold, ACBE-RTS establishes a versatile framework for precision therapeutics and genetic interrogation. Its modular Sddi-Sdd interface could in principle be readily extended to other base editors, such as thymine and guanine base editors (TBE and GBE).},
}

Humans
*Gene Editing/methods
Animals
CRISPR-Cas Systems
CRISPR-Associated Protein 9/genetics/metabolism
Cytosine/metabolism
HEK293 Cells
Adenine/metabolism
Cell Line
DNA, Single-Stranded/metabolism/genetics

@article {pmid41222695,
year = {2026},
author = {Yusuf, M},
title = {CRISPR screens reveal YAP/TEAD axis as a mediator of drug-tolerant cells in EGFR-mutant NSCLC.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {399},
number = {4},
pages = {5761-5776},
pmid = {41222695},
issn = {1432-1912},
mesh = {Humans ; ErbB Receptors/genetics/antagonists & inhibitors ; *Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/metabolism/pathology ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; Drug Resistance, Neoplasm/genetics ; Mutation ; Cell Line, Tumor ; YAP-Signaling Proteins ; *Transcription Factors/genetics/metabolism ; Hippo Signaling Pathway ; *Antineoplastic Agents/pharmacology ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; Acrylamides/pharmacology ; Signal Transduction/drug effects ; Protein Serine-Threonine Kinases/metabolism ; Aniline Compounds/pharmacology ; Gefitinib/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Indoles ; Pyrimidines ; },
abstract = {Despite initial responses, most patients with metastatic lung cancer-including those with EGFR mutations-ultimately develop resistance to targeted therapies. To systematically uncover mechanisms underlying this resistance, genome-wide CRISPR knockout and activation screens were conducted in EGFR-mutant lung cancer cell lines treated with EGFR inhibitors such as osimertinib and gefitinib. These screens highlighted a recurrent involvement of genes associated with the Hippo signaling pathway. Notably, a subset of tumor cells, termed 'persister' cells, survive initial osimertinib exposure by engaging non-genetic, transcriptional adaptation mechanisms that promote drug tolerance. Our studies, integrating both genetic and pharmacological approaches, identified Hippo pathway activation as a key driver of this drug-tolerant state. Importantly, co-inhibition of EGFR and the Hippo signaling axis led to a pronounced reduction in cell viability in both established cell lines and patient-derived organoids. These findings propose that dual targeting of EGFR and Hippo signaling may offer a promising therapeutic approach to overcome resistance in EGFR-mutant lung cancer.},
}

Humans
ErbB Receptors/genetics/antagonists & inhibitors
*Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/metabolism/pathology
*Lung Neoplasms/genetics/drug therapy/metabolism/pathology
Drug Resistance, Neoplasm/genetics
Mutation
Cell Line, Tumor
YAP-Signaling Proteins
*Transcription Factors/genetics/metabolism
Hippo Signaling Pathway
*Antineoplastic Agents/pharmacology
*Adaptor Proteins, Signal Transducing/genetics/metabolism
Protein Kinase Inhibitors/pharmacology
CRISPR-Cas Systems
Acrylamides/pharmacology
Signal Transduction/drug effects
Protein Serine-Threonine Kinases/metabolism
Aniline Compounds/pharmacology
Gefitinib/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
Indoles
Pyrimidines

@article {pmid41411621,
year = {2026},
author = {Tankka, AT and Zhang, Y and Einstein, JM and Zhou, CJ and Pham, VN and Naritomi, JT and Nguyen, GG and Mendez-Molina, AN and Hu, Z and Mizrahi, O and Perelis, M and Sarsam, J and Tan, FE and Kaufman, DS and Yang, J and Antal, CE and Yeo, GW},
title = {Integrative CRISPR Screening and RNA Analyses Discover an Essential Role for PUF60 Interactions with 3' Splice Sites in Cancer Progression.},
journal = {Cancer research},
volume = {86},
number = {7},
pages = {1586-1604},
pmid = {41411621},
issn = {1538-7445},
support = {R01 CA268179/CA/NCI NIH HHS/United States ; S10 OD025060/OD/NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; F32 HL143978/HL/NHLBI NIH HHS/United States ; P30 CA023100/CA/NCI NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; U24 HG011735/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; T32 CA067754/CA/NCI NIH HHS/United States ; P30CA23100//National Cancer Institute (NCI)/ ; S10OD026929//National Institutes of Health (NIH)/ ; 1S10OD025060//National Institutes of Health (NIH)/ ; U54 CA209891/CA/NCI NIH HHS/United States ; T32CA067754//National Institutes of Health (NIH)/ ; //Gruss-Lipper Family Foundation/ ; F32 HL143978/HL/NHLBI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 CA268179/CA/NCI NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; 2023-332369//Chan Zuckerberg Initiative (CZI)/ ; V2024-008//V Foundation for Cancer Research (VFCR)/ ; IRG-19-230-48-IRG//American Cancer Society (ACS)/ ; P30CA023100//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Animals ; Mice ; Female ; Cell Proliferation/genetics ; Disease Progression ; *RNA Splicing Factors/genetics/metabolism ; *CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Apoptosis/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA Splicing ; Xenograft Model Antitumor Assays ; Repressor Proteins ; },
abstract = {UNLABELLED: RNA-binding proteins (RBP) are important regulators of posttranscriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple-negative breast cancer (TNBC). In this study, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated enhanced cross-linking and immunoprecipitation and RNA sequencing analysis identified that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when misspliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3' splice sites via a substitution in its RNA-binding domain caused widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. Knockdown of PUF60 or disruption of PUF60-RNA interactions inhibited TNBC cell proliferation and shrunk tumor xenografts in multiple models. Together, these findings reveal the molecular mechanism by which PUF60 supports cancer progression.

SIGNIFICANCE: Functional screening of RNA-binding proteins is an effective strategy for identifying cancer regulators that revealed PUF60-mediated splicing activity as a driver of oncogenic proliferation and a potential therapeutic target.},
}

Humans
Animals
Mice
Female
Cell Proliferation/genetics
Disease Progression
*RNA Splicing Factors/genetics/metabolism
*CRISPR-Cas Systems
*Triple Negative Breast Neoplasms/genetics/pathology/metabolism
Gene Expression Regulation, Neoplastic
Cell Line, Tumor
Apoptosis/genetics
*RNA-Binding Proteins/genetics/metabolism
RNA Splicing
Xenograft Model Antitumor Assays
Repressor Proteins

@article {pmid41445188,
year = {2026},
author = {Shmuel-Eidelman, M and Cohen-Fultheim, R and Eisenberg, E and Levanon, EY},
title = {Off-target RNA editing hotspots caused by base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2361-2371},
doi = {10.1016/j.ymthe.2025.12.043},
pmid = {41445188},
issn = {1525-0024},
mesh = {*RNA Editing ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Algorithms ; RNA, Guide, CRISPR-Cas Systems/genetics ; Exons ; },
abstract = {Base editors, composed of engineered deaminases fused with Cas proteins and a guide RNA, enable precise, programmable alteration of single nucleotides within the genome and transcriptome. This innovative technology holds promising therapeutic potential for correcting disease-causing point mutations. However, its clinical translation hinges on both high efficacy and accuracy. Non-specific unintended edits by base editors remain a critical challenge. Efforts to mitigate off-target activity have focused mostly on detecting recurrent RNA deaminations at specific sites. Complementarily, our methodology quantifies the total burden of RNA alterations, which is particularly effective for capturing stochastic off-target edits that evade conventional detection. Here, we applied the RNA editing index algorithm to quantify off-target levels across individual genes and identified 2,844 adenine base editors and 1,253 cytosine base editor hotspot genes susceptible to aberrant editing. Exon-level analysis revealed localized regions within genes that are particularly prone to off-target editing, including regions where edits introduce premature stop codons, a critical risk for therapeutic applications. By uncovering these previously unrecognized off-target landscapes, our study deepens our understanding of base editor specificity and provides a framework for optimizing their precision, accelerating the development of safer next-generation editing tools.},
}

*RNA Editing
Humans
*Gene Editing/methods
CRISPR-Cas Systems
Algorithms
RNA, Guide, CRISPR-Cas Systems/genetics
Exons

@article {pmid41472363,
year = {2026},
author = {Ajibaye, O and Dada, IS and Mbye, H and Nwankwo, IC and Oriero, E and Amambua-Ngwa, A},
title = {Innovative approach in malaria research: Harnessing CRISPR-Cas9 for antimalarial drug-resistance studies in Africa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {1909-1920},
doi = {10.1016/j.ymthe.2025.12.034},
pmid = {41472363},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Humans ; *Antimalarials/therapeutic use/pharmacology ; *Drug Resistance/genetics ; Gene Editing/methods ; Africa ; *Plasmodium falciparum/genetics/drug effects ; *Malaria/drug therapy/parasitology/epidemiology/genetics ; Animals ; Malaria, Falciparum/drug therapy/parasitology ; },
abstract = {Malaria remains a major global health challenge, particularly in sub-Saharan Africa, where Plasmodium falciparum is increasingly resistant to frontline antimalarial therapies. CRISPR-Cas9, a groundbreaking genome editing tool, has become a transformative force in biomedical research, offering valuable insights into the genetic mechanisms underlying drug resistance. This review provides a comprehensive overview of the CRISPR-Cas9 system, its origin, evolution, and application in antimalarial drug-resistance research, with particular emphasis on African studies. We examine the role of CRISPR in elucidating resistance pathways, validating molecular markers, and enhancing diagnostic strategies. Additionally, a systematic scoping review highlights the geographic scope and focus of CRISPR-related malaria research conducted across Africa. Significantly low application and adoption were observed. Despite existing technological and regulatory barriers, CRISPR holds strong potential for accelerating antimalarial drug discovery and advancing precision medicine. Its strategic application in malaria-endemic regions could significantly bolster efforts toward disease control and eventual elimination.},
}

*CRISPR-Cas Systems
Humans
*Antimalarials/therapeutic use/pharmacology
*Drug Resistance/genetics
Gene Editing/methods
Africa
*Plasmodium falciparum/genetics/drug effects
*Malaria/drug therapy/parasitology/epidemiology/genetics
Animals
Malaria, Falciparum/drug therapy/parasitology

@article {pmid41485050,
year = {2026},
author = {Wang, K and Li, X and Li, J and Yu, Z and Li, P and Xie, Y and Liu, J and Huang, H and Zhang, S and Zhang, M and Ma, W and Gao, F and Du, X and Wang, J and Capecchi, MR and Wu, S},
title = {Basic enables selection-free efficient knockin of large DNA in primary human T cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2309-2323},
doi = {10.1016/j.ymthe.2025.12.064},
pmid = {41485050},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes/metabolism/immunology ; Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/metabolism ; Animals ; *Gene Knock-In Techniques/methods ; Dependovirus/genetics ; Mice ; *DNA/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Efficient and precise insertion of large DNA fragments into primary human T cells has remained a bottleneck for gene and cell therapy. We present BaEVshort-AAV6 site-specific integration for CAR T (BASIC), a modular platform that combines BaEVshort-pseudotyped virus-like particles for Cas9 RNP delivery with AAV6 donor vectors for homology-directed repair. BASIC achieves >85% knockin efficiency without drug selection or electroporation, preserving cell viability while enabling multiplex genome engineering. Edited chimeric antigen receptor (CAR)-T cells show uniform CAR expression, enhanced cytotoxicity, and complete tumor clearance in vivo. BASIC offers a clinically scalable solution for next-generation cell therapies.},
}

Humans
*T-Lymphocytes/metabolism/immunology
Genetic Vectors/genetics
*Receptors, Chimeric Antigen/genetics/metabolism
Animals
*Gene Knock-In Techniques/methods
Dependovirus/genetics
Mice
*DNA/genetics
Gene Editing
CRISPR-Cas Systems

@article {pmid41494883,
year = {2026},
author = {Mahmood, MA and Greenwood, JR and Millar, AA and Susila, H},
title = {Next-generation genome editing: no transgene, no tissue culture.},
journal = {Trends in plant science},
volume = {31},
number = {4},
pages = {383-385},
doi = {10.1016/j.tplants.2025.12.008},
pmid = {41494883},
issn = {1878-4372},
mesh = {*Transgenes/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Tissue Culture Techniques ; CRISPR-Cas Systems ; },
abstract = {New approaches to engineering plant genomes have the potential to improve agriculture. However, transgenes insertion and tissue culture have become bottlenecks to genome-editing technology becoming widely adopted and achieving the promise of targeted editing. Recent developments in particle bombardment and viral vector-mediated delivery can open doors to overcome these limitations.},
}

*Transgenes/genetics
*Genome, Plant/genetics
Plants, Genetically Modified/genetics
*Gene Editing/methods
*Genetic Engineering/methods
Tissue Culture Techniques
CRISPR-Cas Systems

@article {pmid41526501,
year = {2026},
author = {Garriga-Canut, M and Cannon, N and Benton, M and Zanon, A and Horsfield, ST and Scheurich, J and Remans, K and Lees, J and Paix, A and van Gestel, J},
title = {Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species.},
journal = {Molecular systems biology},
volume = {22},
number = {4},
pages = {599-619},
pmid = {41526501},
issn = {1744-4292},
support = {101116560//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Dictyostelium/genetics ; Gene Knockout Techniques ; Gene Knock-In Techniques ; Phylogeny ; },
abstract = {Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Due to a lack of genome editing methods, most molecular research in Dictyostelids has focused on only a single species, Dictyostelium discoideum, which has severely limited broad-scale comparative analyses. Here, we introduce the first CRISPR-Cas9 editing approach that is cloning-free, selection-free, highly efficient, and effective across Dictyostelid species that diverged millions of years ago. Depending on the CRISPR-Cas9 target site, our editing approach generates knock-out efficiencies of up to 90% and knock-in efficiencies of up to 50% without a selective marker. We show that mutants can be isolated as soon as one day post-transfection, vastly outpacing existing methods for generating knock-outs, fusion proteins, and expression reporters. Leveraging single-cell sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing approach to phylogenetically distant Dictyostelid species, including those that have never been genome edited before. Our methods therefore open the door to performing broad-scale genetic interrogations across the Dictyostelids.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Dictyostelium/genetics
Gene Knockout Techniques
Gene Knock-In Techniques
Phylogeny

@article {pmid41531187,
year = {2026},
author = {Tamauchi, S and Yoshida, K and Xinyuan, W and Nakagawa, A and Yokoi, A and Yoshikawa, N and Niimi, K and Yamamoto, Y and Kajiyama, H},
title = {In-Tumor CRISPR-Cas9 Knockout Screening and Novel Therapy Development for Malignant Transformation of Ovarian Teratoma.},
journal = {Cancer science},
volume = {117},
number = {4},
pages = {983-995},
pmid = {41531187},
issn = {1349-7006},
support = {24K12578//Japan Society for the Promotion of Science/ ; 23ama221125h0001//Japan Agency for Medical Research and Development/ ; },
mesh = {Female ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; *Teratoma/genetics/pathology/drug therapy ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; *Cell Transformation, Neoplastic/genetics/drug effects ; Cell Proliferation/drug effects/genetics ; Apoptosis/drug effects ; Gene Knockout Techniques ; Cisplatin/pharmacology ; Oxidative Stress/drug effects ; },
abstract = {Malignant transformation of mature cystic teratoma (MTMCT) of the ovary is a rare but aggressive malignancy for which no standardized chemotherapy or effective targeted therapies currently exist. To identify therapeutic vulnerabilities in MTMCT, we performed a genome-wide CRISPR-Cas9 knockout screen using the MTMCT-derived NOSCC1 cell line. Two parallel selective pressures were applied: in vivo tumorigenicity in immunodeficient mice and cisplatin exposure in vitro. From this screen, 67 negatively selected genes were identified, among which SOD1 and NDUFB4 emerged as top candidates based on high basal expression levels and clinical relevance. Integration with spatial transcriptomic data from three independent MTMCT patient tumors further supported the prioritization of these targets. SOD1 was selected for further investigation due to the availability of known pharmacological inhibitors. Both siRNA-mediated knockdown and small-molecule inhibition of SOD1 using LCS-1 significantly suppressed MTMCT cell proliferation in vitro by inducing oxidative stress and impairing cell cycle progression. This antiproliferative effect was reversed by co-treatment with N-acetylcysteine, a reactive oxygen species scavenger. In vivo validation using patient-derived xenograft models demonstrated that oral administration of LCS-1 led to significant tumor growth suppression and increased expression of apoptotic and DNA damage markers, including cleaved caspase-3 and γH2AX. These findings establish SOD1 as a critical vulnerability in MTMCT and provide preclinical evidence supporting redox modulation as a therapeutic strategy for this highly chemoresistant and understudied ovarian cancer subtype. Our integrative approach combining functional genomics, spatial transcriptomics, and pharmacologic validation offers a framework for the discovery of novel targets in rare gynecologic malignancies.},
}

Female
*Ovarian Neoplasms/genetics/pathology/drug therapy
*Teratoma/genetics/pathology/drug therapy
Humans
Animals
*CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
Xenograft Model Antitumor Assays
*Cell Transformation, Neoplastic/genetics/drug effects
Cell Proliferation/drug effects/genetics
Apoptosis/drug effects
Gene Knockout Techniques
Cisplatin/pharmacology
Oxidative Stress/drug effects

@article {pmid41536070,
year = {2026},
author = {Zeng, H and Liu, A and Daniel, TC and Golla, DA and Lu, Z and Serodio, R and Millette, BA and Lingineni, A and Gilberd, P and Chee, K and Talloo, K and Peddi, A and Park, SH and Bao, G and Gao, X},
title = {Precision A3G base editors for disease modeling and correction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2343-2360},
pmid = {41536070},
issn = {1525-0024},
support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; R01 HL173243/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; *APOBEC-3G Deaminase/genetics/metabolism ; *Cystic Fibrosis/genetics/therapy ; Mutation ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Cytosine base editors (CBEs) enable efficient cytosine-to-thymine substitutions at targeted genomic loci without introducing double-stranded breaks. Among CBEs, APOBEC3G BEs (A3G-BEs) preferentially edit the second cytosine within a 5'-CC-3' motif in human cells, reducing potential bystander editing. However, A3G-BEs often unintentionally edit multiple CC motifs within their editing window and are limited by protospacer adjacent motif (PAM) constraints imposed by SpCas9, which restricts their applicability. Here, we engineered A3G-BE variants through linker optimization, rational mutagenesis, and the integration of SpG and SpRY Cas9 effectors with relaxed PAM constraints. These improvements enhanced the precision of single-cytosine editing within CC motifs and broadened the targeting scope to previously inaccessible genomic sites. We then validated the engineered A3G-BE variants by precisely installing and correcting cystic fibrosis-causing mutations in HEK293T cells. When applied to 16HBE14o-human bronchial epithelial cells, precise editing modulated cystic fibrosis transmembrane conductance regulator mRNA levels, protein expression, and channel function, establishing precision A3G-BE variants as powerful tools for modeling and treating cystic fibrosis and other human diseases.},
}

Humans
*Gene Editing/methods
HEK293 Cells
CRISPR-Cas Systems
*APOBEC-3G Deaminase/genetics/metabolism
*Cystic Fibrosis/genetics/therapy
Mutation
Cystic Fibrosis Transmembrane Conductance Regulator/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41741438,
year = {2026},
author = {Liu, T and Lin, Y and Liu, Q and Liao, W and Lin, Y and Zhang, Y and Zhang, J and Cao, W and Yang, L and Hong, Z and Lu, Z},
title = {Target-stabilized base editors enable robust high-fidelity RNA editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741438},
issn = {2041-1723},
support = {82327805//National Natural Science Foundation of China (National Science Foundation of China)/ ; KQTD20210811090117032//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {*RNA Editing/genetics ; *Adenosine Deaminase/genetics/metabolism ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; RNA-Binding Proteins/genetics/metabolism ; Mutation ; Gene Editing/methods ; Mice ; Amyotrophic Lateral Sclerosis/genetics ; },
abstract = {RNA base editing using engineered deaminases represents a powerful tool to correct mutations at the RNA level. However, widespread off-target effects, primarily arising from dissociated free deaminases, remain a significant challenge. Here, we devise the RECODE (RNA editing with conditionally stable and enhanced ADAR1 deaminase variants) system, which employs designer degron-tagged ADAR1 deaminase (ADAR1d) with guide RNA (gRNA)-regulated stability. By promoting degradation of gRNA-unbound ADAR1d, RECODE markedly reduces transcriptome-wide edits while maintaining high on-target efficacy. Engineering gRNA for target RNA-induced conformational switching confines ADAR1d stabilization to intended editing sites, further enhancing editing precision. With structure-guided rational engineering of ADAR1d, RECODE efficiently corrects an Amyotrophic Lateral Sclerosis-relevant FUS mutation and installs a therapeutic mutation to Angptl3 in vivo, which mitigate FUS mislocalization to neuronal axons and lower plasma lipids, respectively. These findings establish RECODE as a highly stringent and efficient RNA editing technology and underscore a general principle for enhancing the specificity of RNA-guided protein effectors.},
}

*RNA Editing/genetics
*Adenosine Deaminase/genetics/metabolism
Humans
Animals
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
RNA-Binding Proteins/genetics/metabolism
Mutation
Gene Editing/methods
Mice
Amyotrophic Lateral Sclerosis/genetics

@article {pmid41902262,
year = {2026},
author = {Jones, JE and Gunderson, CE and Wigdahl, B and Nonnemacher, MR},
title = {Breaking into HIV-1's Epigenetic Vault: Cure Strategies to Eliminate the Viral Reservoir.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902262},
issn = {1999-4915},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {*HIV-1/genetics/drug effects/physiology ; Humans ; *Virus Latency/drug effects ; *Epigenesis, Genetic/drug effects ; *HIV Infections/virology/drug therapy/therapy ; Proviruses/genetics/drug effects ; Chromatin ; *Anti-HIV Agents/pharmacology ; CRISPR-Cas Systems ; Animals ; },
abstract = {Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that integrates into the host cell's DNA as a provirus. Transcription from the provirus is regulated in large part by cellular proteins and epigenetic factors. These may be repressive or permissive to productive infection. The host factors that regulate this balance are therefore attractive targets for HIV-1 therapeutics. Indeed, proviral chromatin is the focus of two of the current HIV-1 cure strategies. "Shock and Kill" uses latency reversal agents to open the provirus's chromatin, promoting high levels of gene expression that induce the killing of infected cells. "Block and Lock" uses latency promoting agents to induce heterochromatin, blocking transcription and forcing HIV-1 into a state of deep latency. Here, the compounds investigated in both strategies are reviewed, including their chemical structures, mechanisms of action, and clinical results. Finally, the use of CRISPR-Cas therapeutics and the impact of chromatin architecture on its efficacy are discussed.},
}

*HIV-1/genetics/drug effects/physiology
Humans
*Virus Latency/drug effects
*Epigenesis, Genetic/drug effects
*HIV Infections/virology/drug therapy/therapy
Proviruses/genetics/drug effects
Chromatin
*Anti-HIV Agents/pharmacology
CRISPR-Cas Systems
Animals

@article {pmid41903969,
year = {2026},
author = {Chen, F and Huang, M and Zeng, S and Wan, Y and Qin, A and Yang, X and Fu, D and Negahdary, M and Zhang, C},
title = {A CRISPR/dCas9 mediated electrochemical impedimetric biosensor for sensitive mtDNA detection.},
journal = {Analytica chimica acta},
volume = {1401},
number = {},
pages = {345323},
doi = {10.1016/j.aca.2026.345323},
pmid = {41903969},
issn = {1873-4324},
mesh = {*DNA, Mitochondrial/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; Metal Nanoparticles/chemistry ; Limit of Detection ; Electrodes ; Polymorphism, Single Nucleotide ; Silver/chemistry ; },
abstract = {BACKGROUND: The accumulation of mitochondrial DNA (mtDNA) mutations in cells is closely linked to various human diseases. Detection of single-nucleotide variation (SNV) in mtDNA plays a crucial role in understanding the heteroplasmy of mtDNAs that contain pathogenic changes. While conventional nucleic acid sequencing-based methods are instrumental and complex, which hampered their capability in revealing the extensive diversity of mtDNA. In order to realize trace DNA analysis, recent CRISPR/Cas-based detection methods facilitated with target pre-amplification, while raised the risks of non-specific amplification and cross-carryover contamination. Thus, it is imperative to develop new methods for sensitive and precise SNV detection in mtDNA.

RESULTS: This study developed a CRISPR/dCas9 (deactivated Cas9) mediated electrochemical impedimetric biosensor without target pre-amplification for sensitive and specific detection of SNVs in mtDNA. dCas9/sgRNA complexes were immobilized on the surface of indium tin oxide (ITO) electrode to specifically recognize target mtDNA sequences and initiate hybridization chain reaction (HCR) for signal amplification. Subsequently, positively charged polyethylenimine-coated silver nanoparticles (PEI-Ag NPs) were electrostatically deposited onto the HCR-generated long double-stranded DNA (dsDNA) products, leading to a marked decrease in electrochemical impedance due to the high conductivity of the nanoparticles. The concentration of mtDNA was thus quantified by monitoring the impedance change via electrochemical impedance spectroscopy (EIS). The method could distinguish single- and multi-base mismatches with a low detection limit of 67 fM without pre-amplification. It exhibits excellent anti-interference ability and excellent recovery rates of 90.0% to 108.0% in complex matrices (10% human plasma), and enables accurate mtDNA detection in cell lysates.

SIGNIFICANCE: This free of pre-amplification strategy offers a highly sensitive analytical tool that enables the successfully detection of mtDNA mutation in diverse cells types, and exhibited excellent anti-interference ability in complicated biological specimen. The work presents a viable and promising strategy for the electrochemical detection of cancer-related biomarkers, indicating substantial potential in early clinical diagnosis.},
}

*DNA, Mitochondrial/genetics/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
Metal Nanoparticles/chemistry
Limit of Detection
Electrodes
Polymorphism, Single Nucleotide
Silver/chemistry

@article {pmid41905621,
year = {2026},
author = {Singh, RP and Maity, P and Jaiswal, C},
title = {Genome editing in Parkinson's disease: Unlocking therapeutic avenues through CRISPR-Cas systems.},
journal = {Neurochemistry international},
volume = {196},
number = {},
pages = {106149},
doi = {10.1016/j.neuint.2026.106149},
pmid = {41905621},
issn = {1872-9754},
abstract = {Parkinson's disease (PD) is an illness that causes both motor and non-motor symptoms in the patient which occurs as a result of a progressive loss of dopamine-producing neurons in the substantia nigra. Even though the success of symptomatic treatments is promising, at the same time there is currently no effective therapy that can halt or reverse disease progression. Key genes such as SNCA, LRRK2, and PINK1 are considered as the main hopefuls aspect for the treatment of Parkinson's because mutations of these genes are the reason for the appearance of the familial and sporadic kinds of the disease, respectively. The CRISPR-Cas system, a breakthrough genome-editing technology which enables precise and targeted genetic modifications, renders the possibilities of both PD research and therapy. Examining the mechanics of prime editing, base editing, and CRISPR-Cas9 highlights how effective and precise these methods are for modifying genes. An overview of recent developments in the use of CRISPR to create PD models is also included in the current review, with a focus on the roles these models play in clarifying disease pathways and locating new treatment targets. These models include isogenic cell lines, transgenic animals, and induced pluripotent stem cells (iPSCs). This review highlights the potential of CRISPR-based strategies to correct PD-associated mutations, modulate pathogenic gene expression, and develop neuroprotective interventions targeting key processes such as mitochondrial dysfunction. Furthermore, it critically evaluates the role of CRISPR-based technologies as transformative tools in PD research and therapy while highlighting key challenges for their clinical translation.},
}

@article {pmid41908308,
year = {2026},
author = {Xu, G and Li, S and Li, H and Ren, X and Ding, Y and Pang, X and Liu, X and Tang, Q and Tu, T and Wang, Y and Luo, H and Yao, B and Tian, J and Chen, R and Guan, F},
title = {CasMiner: a deep-learning tool for high-throughput mining and rational design of efficient Cas9.},
journal = {National science review},
volume = {13},
number = {6},
pages = {nwag090},
pmid = {41908308},
issn = {2053-714X},
abstract = {Since its inception, the CRISPR-Cas system, particularly Cas9, has demonstrated immense potential for life science applications, but expansion of the Cas9 toolkit is constrained by sequence-alignment-based strategies for mining and optimization. Here, we developed CasMiner-a deep-learning model for discovering and engineering novel Cas9 proteins. CasMiner achieved 99.63% accuracy in predicting Cas9s and identified VpCas9 from public databases. Experimental validation showed that VpCas9 exhibits robust double-strand cleavage activity. Combining CasMiner and evolutionary analysis, we engineered three mutants with markedly increased structural rigidity and positive charge. In vivo cleavage assays revealed that the mutant VPM2-3 achieved a higher average editing efficiency in rice callus and maize protoplasts than the wild-type VpCas9, the editing efficiency of which rivals that of SpCas9. This study thus establishes a comprehensive platform for mining and engineering Cas9 proteins, and provides VpCas9 and derivative nucleases as powerful tools that greatly broaden the horizon for genome-editing applications.},
}

@article {pmid41909946,
year = {2026},
author = {Zhong, H and Zhou, J and Qin, F and Zhang, XE and Chen, M},
title = {Programmable, target-induced fluorogenic CRISPR-tDeg platform for live-cell RNA visualization.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909946},
issn = {1362-4962},
support = {2023YFA0915603//National Key R&D Program of China/ ; 32101210//National Natural Science Foundation of China/ ; //Young Elite Scientist Sponsorship Program by CAST/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *RNA, Viral/genetics/metabolism ; Fluorescent Dyes/chemistry ; COVID-19/virology ; *RNA ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {RNA molecules display remarkable heterogeneity in structure, dynamics, and function, yet methods for their precise visualization in living cells remain limited. While CRISPR-based RNA imaging holds great potential, existing systems often suffer from high background fluorescence due to constitutive signal emission or non-specific binding. To overcome these challenges, we developed CtDeg (CRISPR-dCas13-tDeg), a modular RNA imaging platform that links fluorescence activation directly to target RNA recognition while leveraging degron-mediated degradation to suppress background signals. By engineering the crRNA scaffold to embed the Pepper RNA motif, CtDeg ensures that fluorescence is present only upon binding to the native RNA target. We systematically optimized C-terminal tDeg variants to maximize the signal-to-noise ratio and demonstrated that CtDeg achieves substantially lower background and higher specificity than conventional fluorescent protein-CRISPR-based RNA imaging approaches. Using CtDeg, we captured real-time paraspeckle assembly dynamics and visualized early-stage SARS-CoV-2 genomic RNA transport. Remarkably, CtDeg provided the first direct imaging evidence of virus-induced NEAT1_2 lncRNA accumulation, revealing a host-virus regulatory interaction. Beyond these applications, CtDeg is compatible with multiple Cas13 orthologs and fluorescent proteins, establishing a versatile, target-induced platform for probing RNA localization, dynamics, and function in living cells, with broad applications in synthetic biology and RNA biology.},
}

Humans
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics
*RNA, Viral/genetics/metabolism
Fluorescent Dyes/chemistry
COVID-19/virology
*RNA
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40744727,
year = {2026},
author = {Sabin, CE and Samudra, SP and Iouchmanov, AL and Rittgers, AL and Lauderdale, JD and Menke, DB},
title = {A Surgical Method for Oocyte Injection and CRISPR-Cas9 Mutagenesis in Anolis Lizards.},
journal = {Cold Spring Harbor protocols},
volume = {2026},
number = {4},
pages = {pdb.prot108652},
pmid = {40744727},
issn = {1559-6095},
support = {R01 HG013006/HG/NHGRI NIH HHS/United States ; T32 GM007103/GM/NIGMS NIH HHS/United States ; T32 GM142623/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Lizards/genetics ; *CRISPR-Cas Systems ; *Oocytes/physiology ; Female ; *Gene Editing/methods ; *Mutagenesis ; *Microinjections/methods ; },
abstract = {Squamates, the taxon that comprises lizards and snakes, are a diverse assemblage of reptiles represented by more than 11,000 described species. Studies of gene function in squamates, however, have remained very limited, largely due to the lack of established genetic tools and suitable experimental systems. A major challenge for the development of CRISPR-based gene editing in these reptiles is that the isolation of fertilized oocytes or single-celled embryos is impractical for most species, given that fertilization occurs internally, the females of many species can store sperm, and simple methods for detecting ovulation are lacking. To overcome these challenges, we have developed a unique surgical approach in the brown anole lizard Anolis sagrei The procedure enables users to access and microinject unfertilized oocytes while they are still maturing within the lizard ovary. We describe here the methods to anesthetize adult female anoles, access the ovary through a surgical incision into the coelomic cavity, and microinject unfertilized oocytes with CRISPR-Cas9 ribonucleoprotein complexes to generate targeted mutations, enabling the routine production of gene-edited lizards.},
}

Animals
*Lizards/genetics
*CRISPR-Cas Systems
*Oocytes/physiology
Female
*Gene Editing/methods
*Mutagenesis
*Microinjections/methods

@article {pmid41342880,
year = {2026},
author = {Kama, Y and Hirano, KI and Masuhara, K and Endo, Y and Suzuki, Y and Fujimoto, M and Matsuda, T and Yahata, T and Kato, M and Hozumi, K and Tanaka, T and Hosokawa, H},
title = {Notch interaction with RUNX factors regulates initiation of the T-lineage program.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {2},
pages = {},
pmid = {41342880},
issn = {1540-9538},
support = {JP19H03692//Japan Society for the Promotion of Science/ ; JP24K22062//Japan Society for the Promotion of Science/ ; JP24K02485//Japan Society for the Promotion of Science/ ; JP23K15307//Japan Society for the Promotion of Science/ ; //Tokai University/ ; //Chugai Foundation for Innovative Drug Discovery Science/ ; //Uehara Memorial Foundation/ ; //Naito Foundation/ ; //Takeda Science Foundation/ ; //SENSHIN Medical Research Foundation/ ; //Princess Takamatsu Cancer Research Fund/ ; //Foundation for Promotion of Cancer Research/ ; //Chemo-Sero-Therapeutic Research Institute/ ; //Vehicle Racing Commemorative Foundation/ ; //Kobayashi Foundation/ ; //Ichiro Kanehara Foundation for the Promotion of Medical Science and Medical Care/ ; //TERUMO Life Science Foundation/ ; //Mitsubishi Foundation/ ; 35-y10//Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics/ ; //Tokai University School of Medicine/ ; //Kyushu University/ ; },
mesh = {*T-Lymphocytes/metabolism/physiology ; *Cell Lineage/genetics ; Gene Knock-In Techniques ; Mice, Transgenic ; *CCCTC-Binding Factor ; *Core Binding Factor Alpha 2 Subunit/metabolism ; CRISPR-Cas Systems ; *Receptor, Notch1/metabolism ; Lymphoid Progenitor Cells/metabolism/physiology ; Mediator Complex/metabolism ; E1A-Associated p300 Protein/metabolism ; Transcriptional Activation/physiology ; Protein Binding ; Protein Domains ; Male ; Female ; Animals ; Mice ; Trans-Activators ; },
abstract = {Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1-binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex reorganization and genomic redeployment in initiating T-lineage program.},
}

*T-Lymphocytes/metabolism/physiology
*Cell Lineage/genetics
Gene Knock-In Techniques
Mice, Transgenic
*CCCTC-Binding Factor
*Core Binding Factor Alpha 2 Subunit/metabolism
CRISPR-Cas Systems
*Receptor, Notch1/metabolism
Lymphoid Progenitor Cells/metabolism/physiology
Mediator Complex/metabolism
E1A-Associated p300 Protein/metabolism
Transcriptional Activation/physiology
Protein Binding
Protein Domains
Male
Female
Animals
Mice
Trans-Activators

@article {pmid41588257,
year = {2026},
author = {Kim, C and Tagmount, A and Zhu, Z and Wilson, F and Li, D and Ostrov, DA and Brad Barbazuk, W and Bacher, R and Vulpe, CD},
title = {Identification of functional genetic components modulating toxicity response to PFOS using genome-wide CRISPR screens in HepG2/C3A cells.},
journal = {Archives of toxicology},
volume = {100},
number = {4},
pages = {1391-1410},
pmid = {41588257},
issn = {1432-0738},
support = {R01ES033625/ES/NIEHS NIH HHS/United States ; R01ES033625/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Fluorocarbons/toxicity ; *Alkanesulfonic Acids/toxicity ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Docking Simulation ; Genome-Wide Association Study ; },
abstract = {Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 µM) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.},
}

Humans
*Fluorocarbons/toxicity
*Alkanesulfonic Acids/toxicity
Hep G2 Cells
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Molecular Docking Simulation
Genome-Wide Association Study

@article {pmid41640336,
year = {2026},
author = {Kim, YJ and Bang, S and Chung, AJ},
title = {Safe and Efficient CRISPR Genome Editing of Primary Human T Cells Using a Droplet-Based Cell Mechanoporation Platform.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {19},
pages = {e12553},
doi = {10.1002/smll.202512553},
pmid = {41640336},
issn = {1613-6829},
support = {2021R1A2C2006224//Korean government (MSIT; Ministry of Science and ICT)/ ; RS-2023-00242443//Korean government (MSIT; Ministry of Science and ICT)/ ; RS-2023-00218543//Korean government (MSIT; Ministry of Science and ICT)/ ; A.J.C//Korean government (MSIT; Ministry of Science and ICT)/ ; },
mesh = {Humans ; *T-Lymphocytes/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Survival ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {T cell engineering is a transformative strategy for adoptive cell therapy, holding the key to treating a wide array of human diseases. However, clinical translation is limited by current intracellular delivery methods that compromise viability, induce stress responses, and restrict scalability. This study presents a microfluidic droplet mechanoporation system tailored for primary human T cells, enabling efficient, stable, and clinically scalable gene delivery. Delivery of 2000 kDa fluorescein isothiocyanate (FITC)-dextran achieves ∼98% efficiency and >90% post-treatment viability, even at high cell densities, supporting the rapid production of therapeutically relevant cell numbers. The platform efficiently delivers mRNA, achieving transfection efficiencies approaching 99%; further, chimeric antigen receptor (CAR)-encoding mRNA is successfully delivered to generate CAR-expressing T cells with tunable surface expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins are effectively delivered for both single and multiplex knockouts (TRAC and PDCD-1), achieving up to a 2.35-fold higher efficiency than electroporation. Longitudinal analyses confirm preserved viability, proliferation, genome integrity, and T cell phenotypic stability. Collectively, these results establish microfluidic droplet mechanoporation as a safe, efficient, and scalable platform for the clinical manufacturing of engineered T cell therapies.},
}

Humans
*T-Lymphocytes/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Cell Survival
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41655242,
year = {2026},
author = {Liu, J and He, N and Cheng, Z and Lin, S and Fu, S and Wang, M and Huang, F and Abou-Elwafa, SF and Al Aboud, NM and Huang, C and Yang, D},
title = {Genetic identification of Pid3-1 and its regulatory role in promoting blast resistance in rice.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {4},
pages = {},
doi = {10.1093/g3journal/jkag027},
pmid = {41655242},
issn = {2160-1836},
support = {2023R1021006//Special Fund for Agro-scientific Research in the Public Interest of Fujian Province/ ; 2024R1022001//Special Fund for Agro-scientific Research in the Public Interest of Fujian Province/ ; CSTB2022TIAD-KPX0018//Chongqing Technology Innovation and Application Development Special Key Project/ ; 32402387//National Natural Science Foundation of China/ ; GJYS202302//National Natural Science Foundation Project Extension Research Project/ ; XTCXGC2021001//5511 Collaborative Engineering Project/ ; },
mesh = {*Oryza/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Plant Proteins/genetics/chemistry/metabolism ; Magnaporthe/pathogenicity ; Chromosome Mapping ; Phenotype ; Genes, Plant ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Alleles ; Ascomycota ; },
abstract = {Rice blast is a destructive rice disease caused by the fungus Magnaporthe oryzae. Here, we identified a resistance gene from the rice cultivar Wanhui 66 which is resistant to the rice blast Guy11 isolate. Genetic mapping positioned a blast resistance locus to chromosome 6. Employing map-based cloning approach ultimately mapped the novel blast resistance locus to a genomic region of 117 kb that contains the Pid3 gene. Candidate gene prediction and cDNA sequencing indicate that the target resistance gene in the Wanhui 66 is allelic to Pid3, thus it was designated Pid3-1. Further analysis showed that the Pid3-1 has 3 nucleotide substitutions, resulting in 3 amino acid substitutions in the Pid3-1 protein, which significantly affect the structure of the Pid3-1 protein as indicated by the 3D structure simulation. The CRISPR/Cas9 system was employed to generate a Pid3-1 knockout mutants that confirmed that the Wanhui 66 resistant phenotype is controlled by Pid3-1. A molecular marker, Indel-6-34, cosegregates with Pid3-1 was identified that could have a great impact on rice breeding against blast disease resistance.},
}

*Oryza/genetics/microbiology
*Disease Resistance/genetics
*Plant Diseases/genetics/microbiology
*Plant Proteins/genetics/chemistry/metabolism
Magnaporthe/pathogenicity
Chromosome Mapping
Phenotype
Genes, Plant
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Alleles
Ascomycota

@article {pmid41687699,
year = {2026},
author = {García, G and Shapiro, JB and Campbell, ZT},
title = {Efficient genetic perturbation of murine sensory neurons in vivo using CRISPR/Cas9.},
journal = {The journal of pain},
volume = {41},
number = {},
pages = {106217},
doi = {10.1016/j.jpain.2026.106217},
pmid = {41687699},
issn = {1528-8447},
mesh = {Animals ; *Sensory Receptor Cells/metabolism ; Ganglia, Spinal/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; *TRPV Cation Channels/genetics/metabolism ; Mice, Transgenic ; NAV1.7 Voltage-Gated Sodium Channel/genetics/metabolism ; Hyperalgesia/genetics/chemically induced ; Sciatic Nerve/metabolism ; *Gene Editing/methods ; Capsaicin ; Male ; Disease Models, Animal ; Mice, Inbred C57BL ; },
abstract = {Gene editing using CRISPR/Cas9 in vivo offers a powerful tool to investigate pain mechanisms. We generated a conditional knock-in mouse model where Streptococcus pyogenes CRISPR-associated protein 9 (Cas9) expression is restricted to cells that express SCN9A. Transgenic markers were detected in key tissues including the dorsal root ganglia (DRG) and sciatic nerve. To assess in vivo editing efficacy, RNA guides targeting TRPV1 were intrathecally administered. Two injections of guide RNAs resulted in a significant reduction of TRPV1 in both the DRG and sciatic nerve without triggering caspase-3-mediated apoptosis or motor deficits. Edited animals exhibited increased withdrawal latencies to heat and reduced nocifensive behaviors following capsaicin injection. Capsaicin-evoked thermal hyperalgesia and mechanical allodynia were diminished. This approach enables rapid and efficient sensory neuron-specific CRISPR/Cas9 gene perturbations for pain research in mice. We envisage that this method can be employed both for the exploration of molecular mechanisms underlying nociception and for the validation of therapeutic targets associated with pain. PERSPECTIVE: There are tremendous opportunities afforded by facile multi-locus genome perturbation of sensory neurons in vivo. This model and approach enables rapid and low-cost genetic depletion experiments in mice.},
}

Animals
*Sensory Receptor Cells/metabolism
Ganglia, Spinal/metabolism
*CRISPR-Cas Systems/genetics
Mice
*TRPV Cation Channels/genetics/metabolism
Mice, Transgenic
NAV1.7 Voltage-Gated Sodium Channel/genetics/metabolism
Hyperalgesia/genetics/chemically induced
Sciatic Nerve/metabolism
*Gene Editing/methods
Capsaicin
Male
Disease Models, Animal
Mice, Inbred C57BL

@article {pmid41735520,
year = {2026},
author = {Panji, JM and Germano, RFV and America, M and Lou, L and Lecordier, L and Tebabi, P and Martin, M and Vanhollebeke, B},
title = {Scalable and multimodal brain angiogenesis and blood-brain barrier genetics by somatic mutagenesis.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41735520},
issn = {2399-3642},
support = {Ctrl-BBB 865176//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Animals ; *Blood-Brain Barrier/metabolism/physiology ; Zebrafish/genetics/embryology ; *Neovascularization, Physiologic/genetics ; Mice ; CRISPR-Cas Systems ; *Mutagenesis ; *Brain/blood supply/metabolism ; Mice, Transgenic ; Angiogenesis ; },
abstract = {The endothelial blood-brain barrier (BBB) is a complex neuroprotective structure whose maturation begins during brain angiogenesis and is completed postnatally. Despite its clinical importance, our understanding of BBB ontogeny and regulation remains limited. This shortcoming results from the difficulty of modeling the BBB in vitro and the current limitation of in vivo BBB genetics to low-throughput Mendelian genetic approaches. In addition, a single animal model facilitating the study of the neurovascular events occurring during both embryogenesis and adulthood is lacking. Here, leveraging the complementary anatomical and experimental attributes of mice and zebrafish, we present an integrated somatic CRISPR-Cas9 gene disruption strategy that allows us to assess candidate genes for their role in brain angiogenesis and BBB permeability within two and six weeks, respectively. Phenotypic assessment of brain angiogenesis is conducted in the transparent embryonic zebrafish hindbrain, while BBB maintenance is evaluated at both the behavioral and the functional level by combining endothelial-specific Cas9 transgenic mice and BBB-targeting adeno-associated viruses. The quick turnaround time and multiplexing capacity of our multi-species approach enable multimodal BBB functional genomics.},
}

Animals
*Blood-Brain Barrier/metabolism/physiology
Zebrafish/genetics/embryology
*Neovascularization, Physiologic/genetics
Mice
CRISPR-Cas Systems
*Mutagenesis
*Brain/blood supply/metabolism
Mice, Transgenic
Angiogenesis

@article {pmid41741655,
year = {2026},
author = {Lei, J and Yang, K and Cao, W and Qi, S and Du, X and Li, H and Wang, Y and Gan, J and Feng, Y and Li, Y and Zhang, W and Bai, B and Lin, X and Su, X and Zhang, Q and Liang, T and Yu, G},
title = {Pancreatic-targeted lipid nanoparticles based on organ capsule filtration.},
journal = {Nature},
volume = {652},
number = {8108},
pages = {220-229},
pmid = {41741655},
issn = {1476-4687},
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Pancreas/metabolism ; Humans ; *Lipids/chemistry ; Pancreatic Neoplasms/therapy/genetics ; RNA, Messenger/genetics/administration & dosage/metabolism ; Female ; Gene Editing ; Male ; Endocytosis ; Cancer Vaccines/immunology/administration & dosage ; Cytokines/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Capsules/chemistry ; Liposomes ; },
abstract = {Achieving pancreatic-targeted delivery marks a breakthrough in treating pancreatic diseases, yet precise delivery remains challenging[1]. Here we identify an explicit and universal principle for pancreatic-selective delivery and propose a pancreatic-targeted lipid nanoparticle (AH-LNP) for mRNA delivery. AH-LNP exhibits size enlargement after assembly with proteins, facilitating capsule-filter-mediated pancreas-selective accumulation and receptor-mediated endocytosis, thereby boosting the pancreatic-targeted ability. Benefiting from this, AH-LNP enables precise and efficient genome editing in the pancreas through the delivery of Cas9 mRNA and single guide RNA (sgRNA), exhibiting promising potential in the treatment of autoimmune pancreatic diseases. Furthermore, pancreatic-targeted delivery of mRNA encoding therapeutic cytokines through AH-LNP demonstrates superior antitumour efficacy when combined with a cancer vaccine or chimeric antigen receptor T cell therapy in multiple pancreatic cancer models. The safety and pancreatic mRNA delivery of AH-LNP were verified in multiple animal models, including non-human primates, demonstrating great promise for clinical translation. Our findings highlight the transformative potential of this pancreatic-targeted mechanism and the derived LNP platform, opening avenues for developing precision therapeutics against diverse pancreatic diseases.},
}

Animals
*Nanoparticles/chemistry
Mice
*Pancreas/metabolism
Humans
*Lipids/chemistry
Pancreatic Neoplasms/therapy/genetics
RNA, Messenger/genetics/administration & dosage/metabolism
Female
Gene Editing
Male
Endocytosis
Cancer Vaccines/immunology/administration & dosage
Cytokines/genetics
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Capsules/chemistry
Liposomes

@article {pmid41742301,
year = {2026},
author = {Lee, YI and Hart, CC and Henley-Beasley, CS and Herr, JS and Zerpa, E and Barton, ER and Hammers, DW and Sweeney, HL},
title = {Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy.},
journal = {Skeletal muscle},
volume = {16},
number = {1},
pages = {},
pmid = {41742301},
issn = {2044-5040},
support = {P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/pathology/physiopathology/genetics/metabolism ; *Muscle, Skeletal/pathology/physiopathology/metabolism ; Rats, Sprague-Dawley ; Disease Models, Animal ; Dystrophin/genetics/metabolism/deficiency ; Rats ; Male ; Muscle Contraction ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats.

METHODS: CRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses.

RESULTS: MDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition.

CONCLUSIONS: MDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.},
}

Animals
*Muscular Dystrophy, Duchenne/pathology/physiopathology/genetics/metabolism
*Muscle, Skeletal/pathology/physiopathology/metabolism
Rats, Sprague-Dawley
Disease Models, Animal
Dystrophin/genetics/metabolism/deficiency
Rats
Male
Muscle Contraction
CRISPR-Cas Systems

@article {pmid41762887,
year = {2026},
author = {Yu, Y and Sun, S and Song, X and Xiong, Z and Song, Z and Peng, C and Zhang, J and Ai, L},
title = {crRNA-engineered CRISPR/Cas12a system coupled with RPA for ultrasensitive detection of Lactiplantibacillus plantarum.},
journal = {Food chemistry},
volume = {509},
number = {},
pages = {148595},
doi = {10.1016/j.foodchem.2026.148595},
pmid = {41762887},
issn = {1873-7072},
mesh = {*CRISPR-Cas Systems ; *Lactiplantibacillus plantarum/genetics/isolation & purification ; Probiotics/analysis ; *Bacterial Proteins/genetics/metabolism ; *RNA, Bacterial/genetics/metabolism ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The growing probiotic industry requires rapid and precise strain detection methods. Here, a one-pot fluorescence platform integrating RPA with an enhanced CRISPR/Cas12a system (termed RPA-ECas12a) was developed for the detection of Lactiplantibacillus plantarum. Through rational 5'end DNA extension of the crRNA, an optimal variant (5'crRNA10) was identified, which increased the trans-cleavage catalytic efficiency of Cas12a by 33% (3.6 × 10[8] M[-1] s[-1]) compared to the wild-type crRNA. The resulting RPA-ECas12a platform detected L. plantarum with a limit of detection of 1.3 CFU/mL, a linear range from 10[1] to 10[7] CFU/mL, and excellent precision (CVs < 10%). The entire detection was completed within 45 min. The platform demonstrated high selectivity and robustness when applied to commercial probiotic powders, yogurts and other complex food matrices. This work not only provides a sensitive and rapid detection tool for probiotic authentication but also offers a generalizable crRNA-engineering strategy to enhance the performance of CRISPR/Cas12a in diagnostic.},
}

*CRISPR-Cas Systems
*Lactiplantibacillus plantarum/genetics/isolation & purification
Probiotics/analysis
*Bacterial Proteins/genetics/metabolism
*RNA, Bacterial/genetics/metabolism
Limit of Detection
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41762997,
year = {2026},
author = {Yu, D and Ren, H and He, P and Li, W and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Stage-aware quantification of the SARS-CoV-2 3CL[pro] biomarker via CsPbBr3@COF-LZU1@AuNP electrochemiluminescence and Cas13a amplification.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129593},
doi = {10.1016/j.talanta.2026.129593},
pmid = {41762997},
issn = {1873-3573},
mesh = {*SARS-CoV-2/enzymology/isolation & purification ; *Biosensing Techniques/methods ; Gold/chemistry ; *COVID-19/diagnosis/virology ; *Metal Nanoparticles/chemistry ; *Electrochemical Techniques/methods ; Humans ; *Luminescent Measurements/methods ; Metal-Organic Frameworks/chemistry ; CRISPR-Cas Systems ; Biomarkers/analysis ; Limit of Detection ; },
abstract = {Direct, activity-based quantification of the SARS-CoV-2 main protease (3CL[pro]) remains challenging in complex matrices. Here we report a water-compatible electrochemiluminescent (ECL) biosensor that integrates a CsPbBr3@COF-LZU1 emitter, a peptide-DNA conformational switch, and CRISPR/Cas13a-assisted amplification to convert protease activity into a robust optical "turn-on" signal. The covalent organic framework physically stabilizes perovskite nanocrystals in aqueous media and, together with a sparse Au nanoparticle layer, supports assembly of ferrocene-terminated reporters that impose an ultralow baseline via near-field/redox quenching. Target-specific cleavage unlocks an initiator that drives entropy-mediated T7 promoter formation and transcription, producing RNA activators that switch on Cas13a collateral cleavage; removal or distancing of ferrocene from the emitter restores photon output. Under optimized conditions the sensor exhibits a broad log-linear response from 10 to 10[8] aM with an ultralow detection limit of 4.31 aM, high analytical selectivity against common interferents, tight fabrication-to-fabrication precision (inter-electrode RSD ∼3%), and practical robustness (≈97% signal retention over 12 h, ≥90% after 7 days at 4 °C, and ∼92% after 120 ECL cycles). Stepwise ECL, cyclic voltammetry, and impedance analysis confirm layer-by-layer assembly and the intended mechanism of ferrocene-mediated quenching and Cas13a-driven recovery. Applied directly to minimally processed pharyngeal swab eluates, the platform resolves cohort-level differences across disease stages and captures the expected attenuation of 3CL[pro] activity in late-stage specimens, supporting stage-aware quantification in real clinical samples. The modular design-reprogrammable at the protease-cleavable motif, promoter template, and crRNA-points to a general route for sensitive, selective, and water-stable ECL assays of enzymatic activity with translational potential.},
}

*SARS-CoV-2/enzymology/isolation & purification
*Biosensing Techniques/methods
Gold/chemistry
*COVID-19/diagnosis/virology
*Metal Nanoparticles/chemistry
*Electrochemical Techniques/methods
Humans
*Luminescent Measurements/methods
Metal-Organic Frameworks/chemistry
CRISPR-Cas Systems
Biomarkers/analysis
Limit of Detection

@article {pmid41833778,
year = {2026},
author = {Quintana, AJ and García-Suárez, R and Prieto, A and Sánchez, J and Gómez, I and Verduzco-Rosas, LA and doNascimento, N and Lopez-Molina, S and Zhang, J and Soberón, M and Bravo, A and Pacheco, S},
title = {Genome editing of ABCB6 transporter confers resistance to cypermethrin in the major pest of corn, Spodoptera frugiperda.},
journal = {Insect biochemistry and molecular biology},
volume = {190},
number = {},
pages = {104537},
doi = {10.1016/j.ibmb.2026.104537},
pmid = {41833778},
issn = {1879-0240},
mesh = {Animals ; *Spodoptera/genetics/drug effects/metabolism/growth & development ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; Gene Editing ; *Pyrethrins/pharmacology ; *Insect Proteins/genetics/metabolism ; *ATP-Binding Cassette Transporters/genetics/metabolism ; CRISPR-Cas Systems ; Bacillus thuringiensis Toxins ; Larva/genetics/growth & development/drug effects/metabolism ; Bacillus thuringiensis ; Hemolysin Proteins ; Bacterial Proteins ; },
abstract = {Spodoptera frugiperda is a major global pest that affect multiple crops, mainly corn and rice. Unfortunately, this pest has evolved resistance to various chemical and biological pesticides. ATP-binding cassette (ABC) transporters, particularly members of the B subfamily, are associated with detoxification by exporting xenobiotics and plant-derived metabolites from the intoxicated insect cells. In addition, some are involved in the mode of action of Bacillus thuringiensis biopesticide Cry toxins, functioning as receptors for these proteins. In this study, we analyzed transcriptomic data from the midgut tissue of S. frugiperda and identified the ABCB6 as one of the most highly expressed transporters within the ABCB subfamily. To explore its functional role, we generated a CRISPR-Cas9 knockout (KO) mutation. Strikingly, loss of SfABCB6 conferred resistance to the chemical pyrethroid insecticide cypermethrin, while the susceptibility to B. thuringiensis Cry1Ab, Cry1Fa and Vip3Aa toxins remained unchanged. Consistently, the ABCB6 CRISPR-Cas9 KO in S. frugiperda derived Sf9 cells conferred resistance to cypermethrin, reiterating the observed larval phenotype. In contrast, the overexpressing of ABCB6 in Sf9 cells exhibited increased susceptibility to cypermethrin. However, SfABCB6 KO showed fitness costs in the insect, as this mutation drastically reduced fertility. Our results provide evidence that SfABCB6 transporter facilitates cypermethrin toxicity participating in insecticide resistance and pointing out its potential role as a novel target for pest management strategies.},
}

Animals
*Spodoptera/genetics/drug effects/metabolism/growth & development
*Insecticide Resistance/genetics
*Insecticides/pharmacology
Gene Editing
*Pyrethrins/pharmacology
*Insect Proteins/genetics/metabolism
*ATP-Binding Cassette Transporters/genetics/metabolism
CRISPR-Cas Systems
Bacillus thuringiensis Toxins
Larva/genetics/growth & development/drug effects/metabolism
Bacillus thuringiensis
Hemolysin Proteins
Bacterial Proteins

@article {pmid41855972,
year = {2026},
author = {Bai, M and Li, Y and Hu, Q and Qing, M and Bai, L},
title = {Enzymatic crRNA stabilization strategy for enhanced CRISPR/Cas12a detection.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129674},
doi = {10.1016/j.talanta.2026.129674},
pmid = {41855972},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; Mycobacterium tuberculosis/genetics/isolation & purification ; Humans ; Influenza A virus/genetics ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases/metabolism/genetics ; RNA Stability ; RNA, Viral/genetics ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system enables precise and rapid nucleic acid recognition through the programmable targeting capability of CRISPR RNA (crRNA). However, the intrinsic instability of crRNA limits the robustness and sensitivity of CRISPR-based molecular diagnosis in practical applications. Herein, we present a simple and general enhancement strategy that suppresses RNase-mediated crRNA degradation. This strategy, termed RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR), leverages RI to specifically prevent crRNA degradation, thereby improving its stability and enhancing the detection performance and anti-interference capability of the CRISPR system. Using influenza A virus (IAV) and mycobacterium tuberculosis (MTB) as model targets, RI-CRISPR improves detection sensitivity by nearly twentyfold compared to conventional CRISPR/Cas12a. Clinical validation using 40 MTB samples, combined with recombinase polymerase amplification (RPA), achieves 100% specificity and 96% sensitivity compared with the GeneXpert assay. Overall, this work provides a practical strategy to enhance sensitivity and robustness of CRISPR-based diagnostics, and is expected to promote further biomedical applications of CRISPR technology.},
}

*CRISPR-Cas Systems/genetics
Mycobacterium tuberculosis/genetics/isolation & purification
Humans
Influenza A virus/genetics
CRISPR-Associated Proteins
*Endodeoxyribonucleases/metabolism/genetics
RNA Stability
RNA, Viral/genetics
Bacterial Proteins

@article {pmid41855973,
year = {2026},
author = {Yang, M and Hu, J and Zhang, Z and Huang, L and Yu, Y and Qiu, D and Zu, Y and Liu, Y and Lin, Z},
title = {Homogeneous electrochemical sensor for sensitive detection of HBV DNA based on magnetic separation and CRISPR/Cas12a protein trans-cleavage.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129676},
doi = {10.1016/j.talanta.2026.129676},
pmid = {41855973},
issn = {1873-3573},
mesh = {*Hepatitis B virus/genetics/isolation & purification ; *DNA, Viral/analysis/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism/chemistry ; Humans ; *Endodeoxyribonucleases/metabolism ; Magnetic Phenomena ; },
abstract = {Infection with hepatitis B virus (HBV) represents a major challenge to global health, especially in areas of inadequate healthcare infrastructure, rapid and on-site detection plays a crucial role in effective disease management and control. Consequently, it is necessary to develop some simple but sensitive HBV screening techniques. In this work, a homogeneous electrochemical biosensor was constructed for sensitive and specific HBV DNA detection through the integration of CRISPR/Cas12a target recognition and magnetic bead-based separation. The sensor exploits the collateral cleavage activity of CRISPR/Cas12a protein upon recognition of target double-stranded DNA, enabling the degradation of methylene blue-labeled single-stranded DNA captured on magnetic beads (MBs). Then the MBs are separated and redissolved in the solution and the electrochemical response of the system can be tested in homogeneous solution. The electrochemical signal has relationship with the concentration of HBV DNA within a range of 10 fM - 10 nM, and the LOD is 3.74 fM. The sensor demonstrates excellent selectivity and biological stability, which has potential application in clinical diagnosis, especially in resource-limited environments.},
}

*Hepatitis B virus/genetics/isolation & purification
*DNA, Viral/analysis/genetics
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
Limit of Detection
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism/chemistry
Humans
*Endodeoxyribonucleases/metabolism
Magnetic Phenomena

@article {pmid41863928,
year = {2026},
author = {Chang, Z and Zhu, B and Wang, Y and Taylor, JA and Dong, H and Zhu, X and Hao, Y and Zhou, Y and Xu, M and Travas-Sejdic, J},
title = {Disposable laser-induced graphene-based biosensor strip for the detection of N-protein using CRISPR activation and ratiometric electrochemical readout.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129667},
doi = {10.1016/j.talanta.2026.129667},
pmid = {41863928},
issn = {1873-3573},
mesh = {*Graphite/chemistry ; *Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; Humans ; *SARS-CoV-2/isolation & purification ; Lasers ; *CRISPR-Cas Systems ; Gold/chemistry ; COVID-19/diagnosis/virology ; Electrodes ; *Coronavirus Nucleocapsid Proteins/analysis ; Metal Nanoparticles/chemistry ; Limit of Detection ; Methylene Blue/chemistry ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; Phosphoproteins ; },
abstract = {Pathogen detection is important for infectious diseases prevention and control. This study presents a novel positive-response, ratiometric electrochemical biosensor constructed on disposable laser-induced graphene (LIG) electrodes. The platform provides dual electrochemical signals for ultrasensitive detection of the SARS-CoV-2 N-protein by harnessing target-activated CRISPR-Cas12a trans-cleavage activity. The developed sensor incorporates a hairpin oligonucleotide (ON) functionalized on gold nanoparticles-deposited LIG electrode surface. Methylene blue (MB) molecules bind to guanine (G) bases of the hairpin ON through inherent affinity, generating the first electrochemical signal. Upon target-induced CRISPR-Cas12a activation, ferrocene (Fc)-labelled indicator ON hybridises with the hairpin ON, displacing the MB molecules and concurrently introduces the Fc as a secondary electrochemical signal reporter. Such displacement triggers a quantifiable decrease in MB electrochemical current and an increase in Fc electrochemical current, generating an Fc/MB ratiometric signal that enhances with increasing target levels and serves as the robust sensor response. The developed ratiometric biosensor achieves a linear response from 0.01 pM to 100 pM of N-protein with a detection limit of 1.3 × 10[-3] pM and with excellent selectivity. The clinical feasibility of the developed ratiometric electrochemical biosensor was confirmed by detecting N-protein in the inactivated cell-cultured SARS-CoV-2. This work demonstrates significant potential in rapid pathogen detection and point-of-care diagnostics.},
}

*Graphite/chemistry
*Biosensing Techniques/methods/instrumentation
*Electrochemical Techniques/methods/instrumentation
Humans
*SARS-CoV-2/isolation & purification
Lasers
*CRISPR-Cas Systems
Gold/chemistry
COVID-19/diagnosis/virology
Electrodes
*Coronavirus Nucleocapsid Proteins/analysis
Metal Nanoparticles/chemistry
Limit of Detection
Methylene Blue/chemistry
Metallocenes/chemistry
Ferrous Compounds/chemistry
Phosphoproteins

@article {pmid41899394,
year = {2026},
author = {Aliev, T and Imatdinov, A and Prudnikova, E and Taranov, O and Emtsova, K and Imatdinov, I and Agafonov, A},
title = {The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles.},
journal = {Current issues in molecular biology},
volume = {48},
number = {3},
pages = {},
pmid = {41899394},
issn = {1467-3045},
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/ ; },
abstract = {Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m-TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m-TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.},
}

@article {pmid41900389,
year = {2026},
author = {Shuang, W and Zeng, X and Li, T and Li, J and Sun, Q and Chen, L},
title = {Screening, Safety Assessment, and Process Optimization of Lactic Acid Bacteria from Traditional Yak Yogurt as Adjunct Cultures.},
journal = {Microorganisms},
volume = {14},
number = {3},
pages = {},
pmid = {41900389},
issn = {2076-2607},
support = {Southwest Minzu University (Grant No. ZYN2025240)//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Cheese ripening is slow and costly, driving interest in accelerating maturation. This study aimed to isolate a safe, efficient adjunct starter from traditional Sichuan yak yoghurt, a niche rich in stress-adapted lactic acid bacteria. From 295 isolates, 15 strains tolerant to high salt, low pH, and low temperature were selected. Using acidification, autolysis, proteolysis, and peptidase activity as indices, principal component analysis identified Limosilactobacillus fermentum 270 as the best candidate. Phenotypic assays showed no haemolysis, gelatin liquefaction, indole production, or amino acid decarboxylase activity. Whole-genome sequencing confirmed species identity and revealed 52 protease/peptidase genes, complete pathways for diacetyl/acetoin biosynthesis and branched-chain amino acid conversion, and no functional biogenic amine synthesis genes. Stress-related genes (F-ATPase, glycine-betaine transport, cold-shock proteins) support cheese adaptability. Antibiotic resistance gene homologs were mainly chromosomal and unlinked to mobile genetic elements; a functional CRISPR-Cas system lowers horizontal transfer risk. The strain was developed as a freeze-dried direct-vat starter (97.3% viability). Orthogonal optimisation of yak Gouda cheese-making defined best conditions: 0.018% adjunct, 45 min acidification, pH 5.8, and 30% curd washing. L. fermentum 270 thus combines proteolytic, flavour-enhancing, genetic safety, and processing traits, offering a promising adjunct for accelerated cheese ripening.},
}

@article {pmid41901428,
year = {2026},
author = {Hou, J and Li, H and Zhang, F and Yang, D and Xiong, Y and Zhu, X and Wen, M},
title = {From Gene Knockouts to Genome Remodeling: Large DNA Fragment Deletion Technologies in Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {41901428},
issn = {2223-7747},
support = {2023YFA0913500//National Key Research and Development Program of China/ ; 25HHWCSS0007//Haihe Laboratory of Sustainable Chemical Transformations/ ; 32230012//the National Natural Science Foundation of China/ ; 32470363//the National Natural Science Foundation of China/ ; },
abstract = {Large DNA fragment deletion (LDFD) provides a powerful means to reconfigure plant genomes at the kilobase to megabase scale, enabling the dissection of genome function, elucidation of non-coding regulatory elements, modulation of gene dosage, reorganization of chromosomal architecture, and implementation of synthetic biology designs. In this review, we systematically compare the mechanisms, efficiencies, advantages, and limitations of the major LDFD technologies that have been applied in plants, including ZFNs, TALENs, CRISPR/Cas systems (Cas9, Cas12a, Cas3), site-specific recombinases, transposon-based systems, and prime editing-derived strategies. We highlight how plant-specific features of chromatin organization and DNA repair constrain large deletions, and discuss the current bottlenecks in achieving efficient, precise, and predictable LDFD across diverse crop genomes. Finally, we outline future directions for plant LDFD, emphasizing AI-assisted design of nucleases and recombinases, protein-directed evolution, and improved DNA- and RNP-based delivery systems. Together, these advances are expected to transform LDFD from a specialized tool into a broadly accessible platform for functional genomics, trait engineering and rational genome design in plants.},
}

@article {pmid41902223,
year = {2026},
author = {Zhang, J and Dang, TT and Lin, TY and Yu, X and Pellin, D and Tian, J and Simmons, O and Kou, E and Cornetta, K and Xiao, W},
title = {Development of a Novel Method to Detect AAV Vector Integration.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902223},
issn = {1999-4915},
support = {P01HL160472//National Institute of Health/ ; 75N92019D00018/HL/NHLBI NIH HHS/United States ; },
mesh = {*Dependovirus/genetics/physiology ; Humans ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Virus Integration ; HeLa Cells ; Nanopore Sequencing ; High-Throughput Nucleotide Sequencing ; },
abstract = {AAV integration has become an important safety consideration in gene therapy. However, accurately determining integration sites remains challenging due to biases introduced by library preparation methods, sequencing technologies, and bioinformatic pipelines. In this study, we developed a PCR-free amplification based on a CRISPR-Cas9 cleavage strategy for AAV DNA that overcomes the limitations of PCR amplification imposed by the ITR structure. When combined with long-read nanopore sequencing, this CRISPR-Cas9-based workflow preserves native AAV integration states and enables unbiased detection of integration junctions. We used AAV-transduced HeLa single-cell clones to evaluate the performance of this approach. To confirm integration site identification, AAV integration junctions were also detected using a probe hybridization capture strategy followed by Illumina short-read sequencing. Integration junctions identified by both methods were further confirmed by PCR. The results showed strong consistency between the two approaches in accurately identifying AAV integration sites in each clone. Overall, these findings demonstrate that the CRISPR-Cas9-enabled, PCR-free long-read sequencing workflow provides a promising tool for characterizing AAV integration events.},
}

*Dependovirus/genetics/physiology
Humans
CRISPR-Cas Systems
*Genetic Vectors/genetics
*Virus Integration
HeLa Cells
Nanopore Sequencing
High-Throughput Nucleotide Sequencing

@article {pmid41902231,
year = {2026},
author = {Qi, M and Liu, X and Wang, W and Lu, M and Zeng, Q and Li, N and Han, Y and Fan, S and Lu, C and Dai, J},
title = {Tree Shrew Genome-Wide CRISPR Screen Identifies RNF6 as a Proviral Host Factor for Zika Virus Replication in Brain Microvascular Endothelial Cells.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902231},
issn = {1999-4915},
support = {2019-1-R-24483//the Bureau of Science and Technology of Kunming/ ; 2017HC019//the Yunnan Science and Technology Talent and Platform Program/ ; D-2024006//the Training Program for High-level Health and Medical Technology Talents in Yunnan Province/ ; },
mesh = {Animals ; *Endothelial Cells/virology ; *Zika Virus/physiology/genetics ; *Virus Replication ; *Brain/virology/blood supply ; Humans ; *Zika Virus Infection/virology/metabolism ; CRISPR-Cas Systems ; *Host-Pathogen Interactions ; Viral Nonstructural Proteins/metabolism ; Blood-Brain Barrier/virology ; },
abstract = {Zika virus (ZIKV), a unique flavivirus with neurotropic and teratogenic potential, can cross the blood-brain barrier and persist in human brain microvascular endothelial cells (BMECs); however, no approved vaccines or specific antivirals exist, and its barrier-crossing and neuroinvasive mechanisms remain elusive. Innovative strategies to identify additional host factors mediating ZIKV infection could yield key insights and help address these challenges. To uncover novel host factors, we established the first tree shrew (Tupaia belangeri) genome-wide CRISPR/Cas9 knockout (GeCKO) library and performed a screen in BMECs, identifying ring finger protein 6 (RNF6) as a novel proviral factor for ZIKV. ZIKV infection in BMECs was significantly reduced following RNF6 knockout or knockdown but enhanced upon RNF6 overexpression or rescue. Mechanistically, RNF6 interacts with the ZIKV NS5 protein and acts as a potential negative regulator of the type I interferon and MAPK signaling pathways. Evolutionary and structural analyses revealed that RNF6 is highly conserved between humans and tree shrews; molecular docking further identified shared NS5-binding residues (Gln-59, Arg-140), supporting the conserved proviral role of human RNF6 in ZIKV infection. Our findings highlight tree shrew GeCKO screening as an efficient approach for identifying novel host factors and establish RNF6 as a critical proviral factor for ZIKV replication in BMECs, providing new insights into ZIKV neurotropic pathogenesis and informing potential antiviral strategies.},
}

Animals
*Endothelial Cells/virology
*Zika Virus/physiology/genetics
*Virus Replication
*Brain/virology/blood supply
Humans
*Zika Virus Infection/virology/metabolism
CRISPR-Cas Systems
*Host-Pathogen Interactions
Viral Nonstructural Proteins/metabolism
Blood-Brain Barrier/virology

@article {pmid41077083,
year = {2025},
author = {Luo, J and Lu, Y and Zhang, Z and Zhang, Q and Shang, Y and Yuan, Y and Duan, X and Zhang, L and Tang, Y and Hu, H and Tong, X and Dai, F},
title = {A conserved Piezo-mRPL2/Jun axis controls reproduction via mitochondrial bioenergetics.},
journal = {Insect biochemistry and molecular biology},
volume = {185},
number = {},
pages = {104417},
doi = {10.1016/j.ibmb.2025.104417},
pmid = {41077083},
issn = {1879-0240},
mesh = {*Mitochondria/physiology ; *Mechanotransduction, Cellular/physiology ; Bombyx ; CRISPR-Cas Systems ; Oxidative Stress ; Phosphorylation ; Transgenes ; Male ; Animals ; Ion Channels ; *Energy Metabolism ; Mice ; *Ribosomal Proteins/metabolism ; *Mitochondrial Proteins/metabolism ; Insect Proteins/metabolism ; Fertility ; },
abstract = {Piezo channels are essential mechanosensors, but their roles in reproduction remain poorly understood. Here, we identify a critical function for BmPiezo in silkworm fertility and embryonic diapause, mediated through a conserved downstream regulatory axis. CRISPR/Cas9-mediated knockout of BmPiezo resulted in male sterility and loss of embryonic diapause, accompanied by dysregulation of ribosome-associated genes and oxidative phosphorylation, and reduced ATP production. We identified mitochondrial ribosomal protein L2 (BmmRPL2) and transcription factor BmJun as key downstream effectors, required for spermatogenesis and diapause, respectively. Homozygous disruption of either gene caused embryonic lethality. Cross-species expression analysis revealed strong expression of Piezo2 and mRPL2 in early human spermatids. Remarkably, transgenic expression of murine Piezo2 in BmPiezo-/-silkworms reactivated the Piezo-mRPL2/Jun axis, restored ATP levels, and rescued reproductive defects. These findings define a conserved pathway that connects mechanical sensing to mitochondrial bioenergetics in reproductive regulation, advancing our understanding of fertility mechanisms across species.},
}

*Mitochondria/physiology
*Mechanotransduction, Cellular/physiology
Bombyx
CRISPR-Cas Systems
Oxidative Stress
Phosphorylation
Transgenes
Male
Animals
Ion Channels
*Energy Metabolism
Mice
*Ribosomal Proteins/metabolism
*Mitochondrial Proteins/metabolism
Insect Proteins/metabolism
Fertility

@article {pmid41664627,
year = {2026},
author = {Hancock, MB and Ruby, DR and Bieler, RA and Cole, DC and Marsden, KC},
title = {Multi-omic analyses identify molecular targets of Chd7 that contribute to CHARGE syndrome model phenotypes.},
journal = {Disease models & mechanisms},
volume = {19},
number = {3},
pages = {},
doi = {10.1242/dmm.052592},
pmid = {41664627},
issn = {1754-8411},
support = {R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; //North Carolina State University/ ; },
mesh = {*CHARGE Syndrome/genetics/pathology ; Animals ; Zebrafish/genetics/metabolism ; Phenotype ; *Zebrafish Proteins/metabolism/genetics ; Disease Models, Animal ; *DNA Helicases/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; *Proteomics ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Mutation/genetics ; Gene Expression Profiling ; Multiomics ; },
abstract = {CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo variants in chromodomain helicase DNA binding protein 7 (CHD7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE syndrome phenotypes. To address this problem, here, we used a previously characterized zebrafish model of CHARGE syndrome to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, we identified that CRISPR/Cas9-mediated knockdown of capgb, nefla or rdh5 phenocopies behavioral defects seen in chd7 mutants, functionally validating the roles of these genes. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome.},
}

*CHARGE Syndrome/genetics/pathology
Animals
Zebrafish/genetics/metabolism
Phenotype
*Zebrafish Proteins/metabolism/genetics
Disease Models, Animal
*DNA Helicases/metabolism/genetics
*DNA-Binding Proteins/metabolism/genetics
*Proteomics
Gene Expression Regulation, Developmental
Gene Knockdown Techniques
Transcriptome/genetics
CRISPR-Cas Systems/genetics
Humans
Mutation/genetics
Gene Expression Profiling
Multiomics

@article {pmid41859774,
year = {2026},
author = {Fang, Z and Hao, Y and Zuo, X and Wang, S},
title = {Target-Gated Ratiometric pH Sensing via Tetrahedral DNA Framework-Based Dual-CRISPR System.},
journal = {Analytical chemistry},
volume = {98},
number = {12},
pages = {9208-9218},
doi = {10.1021/acs.analchem.5c07666},
pmid = {41859774},
issn = {1520-6882},
mesh = {Hydrogen-Ion Concentration ; *DNA/chemistry/metabolism ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Fluorescent Dyes/chemistry ; Extracellular Vesicles/metabolism/chemistry ; },
abstract = {Extracellular vesicle (EV)-mediated communication is tightly regulated by local pH, which governs vesicle biogenesis, cargo release, and membrane fusion. Accurate and context-specific pH sensing is therefore crucial for elucidating EV function and disease-associated microenvironmental regulation. Here, we present a tetrahedral DNA framework (TDF)-orchestrated dual-CRISPR system that integrates orthogonal Cas12a and Cas13a nucleases for target-activated, ratiometric pH detection at lipid membranes. By exploiting the distinct pH-activity profiles of Cas12a (optimal pH ∼ 8.5) and Cas13a (optimal pH ∼ 7.2), combined with their complete substrate orthogonality, we constructed a self-calibrating nanosensor featuring equimolar coassembly of both nucleases and their corresponding fluorogenic reporters at the four vertices of a TDF. The well-defined tetrahedral geometry ensured reproducible molecular organization and stable fluorescence output, eliminating variability inherent to conventional single-fluorophore probes. The sensor exhibited quantitative assembly fidelity and robust pH responsiveness across physiological ranges. Importantly, the Cas module can be programmed for conditional activation, enabling pH sensing only upon recognition of disease-associated biomarkers. Using miR-146a, a regulatory microRNA enriched in EVs implicated in inflammation and cancer progression, as a model target, we demonstrated target-gated pH monitoring on cell-derived exosomes and during liposome fusion events. This work establishes a versatile and generalizable platform for programmable, ratiometric sensing at biomembrane interfaces, offering new opportunities to probe EV-mediated intercellular communication and dynamic microenvironmental regulation.},
}

Hydrogen-Ion Concentration
*DNA/chemistry/metabolism
Humans
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Fluorescent Dyes/chemistry
Extracellular Vesicles/metabolism/chemistry

@article {pmid41897352,
year = {2026},
author = {Atluri, U and Cvammen, W and Kemp, MG},
title = {The Small Molecule SR8278 Inhibits Cell Proliferation Independent of the REV-ERB Nuclear Receptor Proteins in Human Keratinocytes.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897352},
issn = {2218-273X},
support = {GM103583/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Cell Proliferation/drug effects ; *Keratinocytes/drug effects/metabolism/cytology ; *Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism/genetics/antagonists & inhibitors ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Expression Regulation/drug effects ; *Thiophenes/pharmacology ; },
abstract = {The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play important roles in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of different preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. We therefore carried out an RNA-seq analysis and found that genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, and these effects were confirmed at both the RNA and protein level by RT-qPCR and Western blotting, respectively. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce genotoxic stress or apoptosis. Finally, the use of CRISPR/Cas9 genome editing and siRNA-mediated disruption of REV-ERB gene expression showed that the loss of the REV-ERB proteins did not impact the effect of SR8278 on gene expression and cell proliferation. We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and, thus, care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown, particularly in studies involving cell proliferation.},
}

Humans
*Cell Proliferation/drug effects
*Keratinocytes/drug effects/metabolism/cytology
*Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism/genetics/antagonists & inhibitors
CRISPR-Cas Systems
Apoptosis/drug effects
Gene Expression Regulation/drug effects
*Thiophenes/pharmacology

@article {pmid41897386,
year = {2026},
author = {Richter, PR and Graf, J and Haag, FWM and Scudlo, V and Wiesmeth, S and Hauslage, J and Richter, M and Geißler, D and Lebert, M},
title = {Acceleration and Light-Induced Changes in Cytosolic cAMP Concentration in Euglena gracilis.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897386},
issn = {2218-273X},
support = {50WB1128 and 50WB2218)//Deutsches Zentrum für Luft- und Raumfahrt (DLR) BMWi project/ ; },
mesh = {*Cyclic AMP/metabolism ; *Euglena gracilis/metabolism/radiation effects ; *Light ; Adenylyl Cyclases/metabolism/genetics ; *Cytosol/metabolism/radiation effects ; *Acceleration ; Phototaxis ; },
abstract = {The second messenger cyclic AMP (cAMP) is very likely involved in phototactic as well as gravitactic behavior of the unicellular flagellate Euglena gracilis. A slight but significant increase in cAMP was observed when cells encountered sub-threshold acceleration (0.16 × g) force after microgravity [µg]. No differences in cAMP levels were found between cells on a clinostat and 1x-controls. This observation is consistent with the ones of earlier studies. Illumination of cells resulted in a significant increase in cellular cAMP levels. After RNAi-mediated knockdown or CRISPR-Cas9 knockout of the photoactivated adenylyl cyclases PACα and/or PACβ in the photoreceptor, light-induced changes in cAMP levels were no longer observed. In parallel, phototactic behavior was abolished, supporting the essential role of photoactivated adenylyl cyclases in phototaxis. Cells spin around their length axis during locomotion (1-2 Hz). In order to generate a signal in the light direction, the cells should be capable of synthesizing and degrading cAMP within 0.5-1 s. The rapid fixation of cells upon transition from dark to light or light to dark revealed that detectable changes in cAMP-levels (increase or decrease) occur within a 100-200 ms time window, which is sufficiently fast to account for the proposed theoretical kinetics of cAMP oscillations.},
}

*Cyclic AMP/metabolism
*Euglena gracilis/metabolism/radiation effects
*Light
Adenylyl Cyclases/metabolism/genetics
*Cytosol/metabolism/radiation effects
*Acceleration
Phototaxis

@article {pmid41897402,
year = {2026},
author = {Shahannaz, DC and Sugiura, T},
title = {Next-Generation Metabolic Reprogramming in iPSC-Derived Cardiomyocytes: CRISPR-EV Synergy for Precision Cardiac Regeneration.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897402},
issn = {2218-273X},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Regeneration ; Animals ; CRISPR-Cas Systems ; Gene Editing ; Metabolic Reprogramming ; },
abstract = {Cardiovascular disease remains the leading global cause of mortality, largely due to the limited regenerative capacity of adult human myocardium. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a scalable platform for cardiac repair and disease modeling; however, their persistent metabolic immaturity-characterized by reliance on glycolysis, reduced oxidative phosphorylation (OXPHOS), and structurally underdeveloped mitochondria-limits functional integration and long-term therapeutic efficacy. Recent advances indicate that targeted metabolic reprogramming can enhance mitochondrial biogenesis, increase ATP production, and improve stress resilience in iPSC-CMs. This review examines the complementary integration of CRISPR-based metabolic engineering and extracellular vesicle (EV)-mediated metabolic modulation as a systems-level strategy for cardiac maturation. We discuss CRISPR activation, interference, and epigenome-editing approaches targeting regulators such as PGC-1α, TFAM, and PPARs to promote stable enhancement of mitochondrial networks and respiratory capacity. In parallel, engineered EVs delivering miRNAs, metabolic enzymes, and redox modulators provide non-genomic mechanisms to optimize bioenergetic function and mitigate oxidative stress. By synthesizing mechanistic insights, quantitative bioenergetic metrics, and translational considerations, we propose CRISPR-EV synergy as a precision framework for durable metabolic maturation of iPSC-CMs, with implications for regenerative therapy, pharmacologic screening, and myocardial repair.},
}

Humans
*Myocytes, Cardiac/metabolism/cytology
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cellular Reprogramming
*Regeneration
Animals
CRISPR-Cas Systems
Gene Editing
Metabolic Reprogramming

@article {pmid41898355,
year = {2026},
author = {Ye, Y and Huang, L and Fu, H and Wang, J and Jin, Y},
title = {Advances in SRNS Gene Research: From Precision Classification to Precision Diagnosis and Treatment.},
journal = {Biomedicines},
volume = {14},
number = {3},
pages = {},
pmid = {41898355},
issn = {2227-9059},
support = {2024C03211 to Jingjing Wang//Key Research and Development Program of Zhejiang Province/ ; GZY-KJS-ZJ-2026-071//Joint TCM Science &Technology Projects of National Demonstration Zones for Comprehensive TCM Reform/ ; LTGD24H050002//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {To clarify the genetic classification, diagnostic strategies, and precision treatment pathways of steroid-resistant nephrotic syndrome (SRNS), this review systematically reviews the genetic stratification system of SRNS by integrating recent advances in genetic testing technologies and pathogenesis research. It contains the pathogenic mechanisms, diagnostic protocols, and therapeutic correlations of different genetic subtypes, while summarizing current progress and clinical challenges in gene therapy. Results indicate SRNS can be categorized into genetic (38-58%) and non-genetic/immune-mediated (40-60%). A stepwise diagnostic system comprising core proteinuria gene panel testing, whole-genome sequencing (WGS), whole-exome sequencing (WES), and supplementary multi-omics/long-range sequencing is proposed, suited for populations with "typical phenotypes and moderate genetic risk", "atypical phenotypes and high genetic suspicion", and "complex structural/non-coding region variants" respectively. Pathogenic mechanisms directly determine therapeutic strategies: COQ2/PDSS2 mutations respond to coenzyme Q10 suplementation, while NPHS1 mutations necessitate early renal transplantation. Adeno-associated virus (AAV)-mediated gene therapy and CRISPR-Cas editing show preclinical promise but face challenges including incomplete detection coverage and clinical translation difficulties. Genetic technologies are driving SRNS management transformation from "empirical treatment" to "mechanism-oriented precision diagnosis and therapy". Future efforts should focus on overcoming genetic testing limitations and gene therapy translation bottlenecks to enhance diagnostic and therapeutic efficacy.},
}

@article {pmid41898529,
year = {2026},
author = {Thu, KL and Jafari, S and Silvester, J and Cruickshank, J and Soria-Bretones, I and Hodgson, K and Tobin, C and Haight, J and Lau, APY and Bray, T and Wakeham, D and Bray, MR and Mak, TW and Cescon, DW},
title = {Genome-Wide CRISPR Screens Identify ABCG2-Mediated Drug Resistance to the Threonine Tyrosine Kinase (TTK) Inhibitor CFI-402257 in Breast Cancer.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898529},
issn = {1422-0067},
mesh = {Humans ; *ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism ; *Drug Resistance, Neoplasm/genetics ; Animals ; Female ; Cell Line, Tumor ; Mice ; *Triple Negative Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; *Neoplasm Proteins/genetics/metabolism ; Xenograft Model Antitumor Assays ; *Protein-Tyrosine Kinases ; CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology ; *Cell Cycle Proteins/antagonists & inhibitors ; *Indoles/pharmacology ; Antineoplastic Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Apoptosis/drug effects ; Protein Serine-Threonine Kinases ; },
abstract = {CRISPR screens are a powerful functional genomics approach for identifying genes that confer sensitivity and resistance to anti-cancer therapies. CFI-402257 (luvixasertib, 2257) is a small molecule inhibitor of threonine tyrosine kinase (TTK), a promising therapeutic target in genomically unstable cancers due to its critical role in establishing the spindle assembly checkpoint (SAC) during mitosis. To inform its ongoing development and evaluation in clinical trials, we sought to use CRISPR activation (i.e., gain of function) screens to identify cellular mechanisms of resistance to 2257 in models of triple-negative breast cancer (TNBC). In vitro screens conducted in two TNBC cell lines nominated ABCG2 as the top resistance-conferring gene in both models. Validation studies assessing clonogenic survival and apoptosis confirmed that ABCG2 overexpression enhanced TNBC resistance to 2257 in vitro, while knockdown enhanced sensitivity. These findings suggest that 2257 is a substrate of ABCG2's drug efflux activity. However, overexpression of ABCG2 failed to confer resistance to 2257 in TNBC xenografts grown in mice and treated with a moderately active dose and schedule. Our results highlight the potential impact of drug transporters in in vitro CRISPR screens and the importance of confirming the relevance of drug response mechanisms identified in cultured cells using in vivo models that recapitulate drug pharmacokinetics and pharmacodynamics.},
}

Humans
*ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism
*Drug Resistance, Neoplasm/genetics
Animals
Female
Cell Line, Tumor
Mice
*Triple Negative Breast Neoplasms/genetics/drug therapy/pathology/metabolism
*Neoplasm Proteins/genetics/metabolism
Xenograft Model Antitumor Assays
*Protein-Tyrosine Kinases
CRISPR-Cas Systems
*Protein Kinase Inhibitors/pharmacology
*Cell Cycle Proteins/antagonists & inhibitors
*Indoles/pharmacology
Antineoplastic Agents/pharmacology
Clustered Regularly Interspaced Short Palindromic Repeats
Apoptosis/drug effects
Protein Serine-Threonine Kinases

@article {pmid41898703,
year = {2026},
author = {Seh, BA and Rafiq, K and Legradi, A and Mir, MY},
title = {Targeted Gene and Genome-Editing Strategies for Epilepsy: Experimental Advances and Translational Challenges.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898703},
issn = {1422-0067},
mesh = {Humans ; *Epilepsy/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; },
abstract = {Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies.},
}

Humans
*Epilepsy/genetics/therapy
*Gene Editing/methods
*Genetic Therapy/methods
Animals
CRISPR-Cas Systems
Translational Research, Biomedical

@article {pmid41898716,
year = {2026},
author = {Calbay, O and Hsieh, CL and Lu, C and Ghosh, S and Vijaykumar, V and Watts, I and Sweigard, H and Gandhi, J and den Hollander, AI},
title = {Mapping the Hypoxic Fitness Landscape of Retinal Pigment Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898716},
issn = {1422-0067},
mesh = {*Retinal Pigment Epithelium/metabolism/cytology ; Humans ; Cell Hypoxia/genetics ; Gene Expression Profiling ; Cell Line ; Transcriptome ; Macular Degeneration/genetics/metabolism/pathology ; CRISPR-Cas Systems ; *Epithelial Cells/metabolism ; Mitochondria/metabolism ; *Hypoxia/genetics ; Oxygen/metabolism ; },
abstract = {Chronic hypoxia is a hallmark of aging and retinal diseases such as age-related macular degeneration (AMD), yet the molecular mechanisms that enable retinal pigment epithelium (RPE) cells to survive under sustained low-oxygen conditions remain poorly understood. To address this, we conducted transcriptomic profiling and a genome-wide CRISPR-Cas9 loss-of-function screen in ARPE-19 cells exposed to chronic hypoxia (1% and 5% O2), mimicking the retinal disease environment. The CRISPR screen identified genes whose loss compromises RPE viability or fitness under hypoxia, while transcriptomic profiling revealed oxygen-dependent shifts in key functional modules. These findings converged on pathways related to mitochondrial function, extracellular matrix remodeling, vascular signaling, and cell cycle regulation, identifying unique functional nodes specific to RPE cells. These core processes are also implicated in retinal diseases, such as AMD. Together, these complementary approaches provide an integrated view of the molecular networks driving RPE adaptation to hypoxic stress and highlight novel gene candidates that may serve as therapeutic targets in retinal disease.},
}

*Retinal Pigment Epithelium/metabolism/cytology
Humans
Cell Hypoxia/genetics
Gene Expression Profiling
Cell Line
Transcriptome
Macular Degeneration/genetics/metabolism/pathology
CRISPR-Cas Systems
*Epithelial Cells/metabolism
Mitochondria/metabolism
*Hypoxia/genetics
Oxygen/metabolism

@article {pmid41898816,
year = {2026},
author = {Abdalla Elsayed, MEA and MacLaren, RE},
title = {Precision Is Not Enough: When Tools Outpace Translation in Ocular Gene Therapy.},
journal = {Genes},
volume = {17},
number = {3},
pages = {},
pmid = {41898816},
issn = {2073-4425},
support = {//Foundation Fighting Blindness Clinical Research Fellowship/ ; //Oxford NIHR Biomedical Research Centre, UK Department of Health/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; Gene Editing/methods ; *Eye Diseases/therapy/genetics ; CRISPR-Cas Systems ; Translational Research, Biomedical ; Animals ; },
abstract = {Advances in molecular biology have positioned the eye as a leading platform for gene therapy, owing to its surgical accessibility, relative immune privilege, and the ability of the contralateral eye to serve as an anatomical control. We trace the historical evolution of gene discovery, synthesize current gene therapy strategies for inherited and acquired ocular disorders, critically evaluating the limitations of CRISPR and related genome-editing technologies, and examine the key scientific and translational challenges that must be addressed for genetic therapies to be integrated into routine ophthalmic practice.},
}

Humans
*Genetic Therapy/methods
Gene Editing/methods
*Eye Diseases/therapy/genetics
CRISPR-Cas Systems
Translational Research, Biomedical
Animals

@article {pmid40148722,
year = {2026},
author = {Mete, R and Das, S and Saha, A and Roy, S and Mondal, S and Bose, A and Basu, B and Elossaily, GM and Prajapati, B},
title = {Transgenesis in Drug Discovery: Enhancing Target Identification and Validation.},
journal = {Molecular biotechnology},
volume = {68},
number = {3},
pages = {998-1022},
pmid = {40148722},
issn = {1559-0305},
mesh = {Animals ; *Drug Discovery/methods ; Humans ; Animals, Genetically Modified/genetics ; *Gene Transfer Techniques ; Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; },
abstract = {Transgenesis, the introduction of foreign genetic material into the genome of an organism, has become a crucial and transformative technique in the realm of drug discovery. This review article provides a comprehensive overview of the integral role that transgenesis plays in the drug discovery process, with a specific focus on target identification and target validation. By examining the recent advancements and innovative approaches, this article aims to shed light on the importance of transgenesis in accelerating drug development. In the context of target identification, transgenesis has allowed for the creation of relevant disease models, enabling researchers to study the genetic and molecular basis of various disorders. The use of transgenic animals, such as mice and zebrafish, has facilitated the identification of potential drug targets by mimicking specific human disease conditions. This review also discusses emerging technologies, such as CRISPR-Cas9 and other genome editing tools, which have revolutionized the field of transgenesis. These technologies have enhanced the precision and efficiency of genetic manipulations in transgenic animals, making the creation of disease-relevant models more accessible and cost-effective. Moreover, integration of omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, has provided a holistic view of the molecular changes in transgenic models, further aiding in target identification and validation. This review emphasizes the importance of transgenesis in target identification and validation and underscores its vital role in shaping the future of drug discovery.},
}

Animals
*Drug Discovery/methods
Humans
Animals, Genetically Modified/genetics
*Gene Transfer Techniques
Gene Editing/methods
CRISPR-Cas Systems
Mice
Disease Models, Animal

@article {pmid40493161,
year = {2026},
author = {Kurpejović, E and Sariyar Akbulut, B and Avci, FG},
title = {Tailoring Corynebacterium glutamicum for Sustainable Biomanufacturing: From Traditional to Cutting-Edge Technologies.},
journal = {Molecular biotechnology},
volume = {68},
number = {3},
pages = {1106-1126},
pmid = {40493161},
issn = {1559-0305},
mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Metabolic Engineering/methods ; Machine Learning ; Genetic Engineering/methods ; CRISPR-Cas Systems ; Artificial Intelligence ; Amino Acids/biosynthesis ; Industrial Microbiology/methods ; Genome, Bacterial ; Biotechnology/methods ; },
abstract = {As the workhorse of industrial amino acid production, Corynebacterium glutamicum is the focus of this review, which provides a comprehensive overview of available techniques employed to engineer strains with desired traits. The review highlights both traditional and cutting-edge approaches with a brief introduction to the bacterium's physiology, serving as a foundation for understanding its metabolic capabilities and potential applications. Genome modulation techniques by contrasting traditional methods with CRISPR-based approaches, as well as transcription modulation strategies that enhance gene expression and metabolic flux, and high-throughput techniques that streamline strain development processes are summarized. Furthermore, the roles of artificial intelligence and machine learning in genetic engineering are explored, emphasizing their growing impact on strain development.},
}

*Corynebacterium glutamicum/genetics/metabolism
*Metabolic Engineering/methods
Machine Learning
Genetic Engineering/methods
CRISPR-Cas Systems
Artificial Intelligence
Amino Acids/biosynthesis
Industrial Microbiology/methods
Genome, Bacterial
Biotechnology/methods

@article {pmid41723140,
year = {2026},
author = {Shen, K and Seow, WY and Keng, CT and Lim, MGL and Lim, DS and Guo, K and Meliani, A and Irfan Bin Hajis, M and Wang, B and Prabhakar, S and Chen, KH and Chew, WL},
title = {Spatial perturb-seq: single-cell functional genomics within intact tissue architecture.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41723140},
issn = {2041-1723},
support = {PREPARE-OC-VT-2024-008//Ministry of Health -Singapore (MOH)/ ; SC18/21-1089UI//Agency for Science, Technology and Research (A*STAR)/ ; OFIRG24jul-0096//MOH | National Medical Research Council (NMRC)/ ; OFYIRG23jul-0050//MOH | National Medical Research Council (NMRC)/ ; NRF-CRP25-2020-0001//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; },
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *Genomics/methods ; Brain/metabolism/cytology ; CRISPR-Cas Systems ; Neurodegenerative Diseases/genetics ; Mice, Knockout ; Neurons/metabolism ; Cell Communication/genetics ; Gene Knockout Techniques ; },
abstract = {We develop Spatial Perturb-Seq, an in vivo CRISPR technology that interrogates multiple genes within single cells of intact tissues, compatible with both sequencing-based and probe-based spatial technologies. We apply Spatial Perturb-Seq to knock out risk genes for neurodegenerative diseases in the mouse brain, uncovering cell autonomous and cell-cell microenvironmental effects within the spatially intact tissue. Spatial Perturb-Seq functionally screens multiple genes in situ and in vivo, bypasses cell processing steps that skew cell type representation, identifies intracellular and intercellular effects of knockouts, and identifies candidate genes underlying dysregulated neuronal intercellular communication pathways.},
}

Animals
*Single-Cell Analysis/methods
Mice
*Genomics/methods
Brain/metabolism/cytology
CRISPR-Cas Systems
Neurodegenerative Diseases/genetics
Mice, Knockout
Neurons/metabolism
Cell Communication/genetics
Gene Knockout Techniques

@article {pmid41840760,
year = {2026},
author = {Karchner, SI and Aluru, N and Franks, DG and Mandl, CA and Goldstone, JV and Burke, T and Champlin, D and La Du, JK and Perone, DM and Stinson, S and Truong, L and Clark, BW and Nacci, D and Tanguay, RL and Hahn, ME},
title = {Using fish models to understand the role of aryl hydrocarbon receptor (AHR)-interacting protein (AIP) in controlling sensitivity and resistance to dioxin-like compounds in vivo.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {209},
number = {3},
pages = {},
doi = {10.1093/toxsci/kfag033},
pmid = {41840760},
issn = {1096-0929},
support = {P42 ES007381/ES/NIEHS NIH HHS/United States ; R01 ES032323/ES/NIEHS NIH HHS/United States ; P30 ES030287/ES/NIEHS NIH HHS/United States ; S10 OD032203/OD/NIH HHS/United States ; R01 ES033888/ES/NIEHS NIH HHS/United States ; P42 ES016465/ES/NIEHS NIH HHS/United States ; R01ES033888//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; R01ES032323//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES007381//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES016465//Superfund Research Program at Boston University/ ; P30ES030287//Superfund Research Program at Oregon State University/ ; 1S10OD032203-01//Superfund Research Program at Oregon State University/ ; //Tufts University Core Facility Genomics Core/ ; //NIH/ ; //NIH Public Access Policy/ ; //PubMed Central upon the Official Date of Publication/ ; },
mesh = {Animals ; *Receptors, Aryl Hydrocarbon/metabolism/genetics ; *Fundulidae/genetics/embryology/metabolism ; *Dioxins/toxicity ; Polychlorinated Dibenzodioxins/toxicity ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Polychlorinated Biphenyls/toxicity ; Zebrafish/genetics ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics ; Embryo, Nonmammalian/drug effects ; *Fish Proteins/genetics/metabolism ; },
abstract = {Humans are exposed to chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) that cause toxicity through activation of the aryl hydrocarbon receptor (AHR). There is inter-individual variation in sensitivity to the effects of AHR ligands, but it is not fully explained by variation in the AHR. A clue to the genetic mechanisms underlying differential sensitivity to AHR agonists has emerged from studies of Atlantic killifish (Fundulus heteroclitus) populations with evolved tolerance to PCBs, TCDD, and PAHs. Genomic studies of these populations identified AHR-interacting protein (AIP/Ara9/XAP2) as the strongest candidate resistance gene. However, the precise role of AIP in the mechanism of resistance is unknown. To understand the role of AIP in the toxicity of dioxin-like compounds in vivo, we used CRISPR-Cas9 to generate AIP loss-of-function alleles in killifish and zebrafish (Danio rerio). Homozygous mutant killifish and zebrafish die during larval development-by 30 and 12 d postfertilization, respectively-whereas heterozygous mutants develop, survive, and reproduce normally. During embryonic and early larval stages, homozygous mutant zebrafish exhibit reduced sensitivity to embryotoxic effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) and TCDD. Gene expression profiling of aip-deficient larvae revealed hundreds of differentially expressed genes. PCB126 induced similar sets of well-known AHR-regulated genes in mutant and wild-type larvae, although with reduced magnitude overall in AIP mutants. This study highlights the important role of AIP in fish larval development and demonstrates that AIP status can influence the response of vertebrate embryos to dioxin-like compounds in vivo.},
}

Animals
*Receptors, Aryl Hydrocarbon/metabolism/genetics
*Fundulidae/genetics/embryology/metabolism
*Dioxins/toxicity
Polychlorinated Dibenzodioxins/toxicity
*Intracellular Signaling Peptides and Proteins/genetics/metabolism
Polychlorinated Biphenyls/toxicity
Zebrafish/genetics
CRISPR-Cas Systems
Zebrafish Proteins/genetics
Embryo, Nonmammalian/drug effects
*Fish Proteins/genetics/metabolism

@article {pmid41892062,
year = {2026},
author = {Tantai, W and Xu, Q and Zhang, W and Li, Y and Liu, H},
title = {A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection.},
journal = {Biosensors},
volume = {16},
number = {3},
pages = {},
pmid = {41892062},
issn = {2079-6374},
support = {22074085//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; *Biosensing Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10[6] copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples.},
}

*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Polymerase Chain Reaction
*Biosensing Techniques
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41892280,
year = {2026},
author = {Woronkowicz, M and Thomas, MN and Saram, SJ and Carr, AF and Alonso-Carriazo Fernandez, A and Butt, Z and Skopiński, P and Ramsden, CM},
title = {CRISPR and Beyond: Genome-Editing Strategies in Retinal Stem Cell Research.},
journal = {Cells},
volume = {15},
number = {6},
pages = {},
pmid = {41892280},
issn = {2073-4409},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Retina/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Stem Cell Research ; Retinal Degeneration/genetics/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing has emerged as a transformative approach for understanding and treating retinal degenerative diseases. Combining this technology with pluripotent stem cells provides an ideal platform for modeling human development and disease, and investigating emerging therapeutic strategies ultimately aimed towards in vivo correction. This approach enables both functional studies to understand retinal degeneration and the early development of targeted therapies for inherited disease. This review offers a comprehensive overview of genome-editing techniques and the ability to create new clinically relevant models to understand human disease in retinal research, focusing on the use of the CRISPR-Cas9 system in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), as well as highlighting recent advancements in base and prime editing. Gene editing in various retinal diseases is discussed in context of studies focusing on disease modeling or developing therapeutic strategies. Continued refinement of these techniques will be essential for advancing translational applications in retinal disease treatment.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
*Retina/cytology
Induced Pluripotent Stem Cells/metabolism/cytology
*Stem Cell Research
Retinal Degeneration/genetics/therapy
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41892424,
year = {2026},
author = {Gomes, E and Mesquita, TG and Serra, P and Araújo, D and Almeida, C and Machado, A and Oliveira, R and Castro, J},
title = {Antimicrobial Resistance in the Food Chain: Bridging Knowledge Gaps for Effective Detection and Control.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41892424},
issn = {2079-6382},
support = {https://doi.org/10.54499/2024.13640.PEX//Fundação para a Ciência e Tecnologia/ ; https://doi.org/10.54499/2022.07654.PTDC//Fundação para a Ciência e Tecnologia/ ; APTA4shiga (number 14840)//Fundação para a Ciência e Tecnologia/ ; },
abstract = {Antimicrobial resistance (AMR) poses a critical global public health threat, with the food chain serving as a significant transmission route connecting animals, environment, and humans. This review adopts a One Health perspective to analyze the key drivers of AMR dissemination across animal agriculture, aquaculture and food processing. We evaluate detection methodologies, contrasting the regulatory gold standard of culture-based phenotypic testing with rapid molecular advancements, including Whole Genome Sequencing (WGS), metagenomics, and emerging CRISPR-Cas diagnostics. While molecular tools offer unprecedented speed and resolution, challenges such as matrix interference, the viable but non-culturable (VBNC) state, and the genotype-phenotype disconnect remain. Finally, integrated mitigation strategies are also described, ranging from on-farm antimicrobial stewardship and innovative biofilm control to consumer hygiene practices. It is essential to bridge the technical and regulatory gaps in AMR surveillance in order to develop effective interventions and ensure a safer food system.},
}

@article {pmid41893903,
year = {2026},
author = {Baliyan, N and Upadhyay, P and Murugan, T and Srivastava, A and Singh, S and Tomar, BS and Mangal, M},
title = {Recent advances in generation of doubled haploid plants for genetic improvement in solanaceous vegetable crops.},
journal = {Planta},
volume = {263},
number = {5},
pages = {},
pmid = {41893903},
issn = {1432-2048},
support = {SR/WOS-A/LS-146/2019//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Haploidy ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; *Vegetables/genetics ; Genome, Plant ; },
abstract = {Genotype-specific protocols and advancements focusing on CRISPR/Cas9-based haploid induction for doubled haploid (DH) production are poised to revolutionize plant breeding for faster genetic improvement in solanaceous crops. The need for swift development of improved cultivars offering greater resilience to biotic and abiotic stresses stems from the emergence of climate change risks. While conventional methods are effective, novel methods for precise crop genome manipulation are required. The in vivo and in vitro protocols leading to fixation of homozygosity and rapid attainment of homozygous DH (doubled haploids) lines have led to a resurgence in research on haploids and DH. The efforts for haploid production have been primarily concentrated on in vitro androgenesis and gynogenesis. The success of these in vitro techniques depends on various parameters, including culture conditions, developmental stage of the microspore, pretreatment, culture medium, growth regulators, and other media additives. Breakthroughs in genome-editing technologies, such as the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, have provided a new avenue for exploring in vivo haploid embryogenesis for haploid induction by means of haploid inducer factors, such as DMP (DMP domain of unknown function 679 membrane protein), ECS (egg cell-specific), MTL (matrilineal), and BBM (Baby boom). A successful haploid induction system in vegetable crops will be possible through the development of efficient in vivo and in vitro androgenesis and gynogenesis protocols. For genome-editing to be feasible, protocols must be optimized for commercially significant solanaceous crops like pepper, potatoes, eggplant, and tomatoes. The existing protocols for genome doubling need to be further improved for solanaceous crops. These developments are pivotal for the advancement and harnessing of haploid technology to its full potential in crop breeding.},
}

*Haploidy
*Crops, Agricultural/genetics
*Plant Breeding/methods
Gene Editing/methods
CRISPR-Cas Systems
*Vegetables/genetics
Genome, Plant

@article {pmid41893914,
year = {2026},
author = {Pistone, D and Bevivino, G and Dipaola, MG and Bandi, C and Lombardo, F},
title = {Current and emerging molecular diagnostic approaches in the detection of human parasites.},
journal = {Parasitology research},
volume = {125},
number = {1},
pages = {},
pmid = {41893914},
issn = {1432-1955},
abstract = {Microscopy and morphological identification remain the gold standard for diagnosing most parasitic infections, yet their limited sensitivity in asymptomatic or low-burden cases, along with technical constraints, has accelerated the adoption of molecular diagnostics. Over the past three decades, advances in nucleic acid amplification and sequencing technologies have transformed parasite detection by improving sensitivity, specificity, and reproducibility, enabling earlier intervention and stronger surveillance. PCR remains the foundation of molecular diagnostics, with real-time PCR and digital PCR improving analytical performance and quantification. Multiplex qPCR supports simultaneous detection of multiple pathogens, while dPCR enables absolute quantification and rare variant detection, although broader implementation is limited by instrument cost. Isothermal amplification methods such as tHDA, NASBA, LAMP, and RPA offer rapid, low-cost amplification at constant temperature and are well suited for field diagnostics in resource-limited settings. Next-Generation Sequencing has advanced genotyping and epidemiological surveillance by resolving cryptic species, resistance mutations, and mixed infections through targeted panels, whole-genome sequencing, and metagenomics. CRISPR/Cas-based assays provide rapid and sensitive nucleic acid detection with strong potential for point-of-care deployment due to their simplicity and adaptability. Emerging biomarkers, including circulating cell-free DNA, non-coding RNAs, and microRNAs in extracellular vesicles, offer promising non-invasive diagnostic strategies, though further validation is required. This review offers a concise overview of these molecular approaches, emphasizing recent innovations such as dPCR, NGS, CRISPR/Cas systems, and biomarker-based detection. For each method, core technical principles, representative applications, and comparative strengths and limitations are presented to illustrate their diagnostic potential.},
}

@article {pmid41895443,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111401},
doi = {10.1016/j.jbc.2026.111401},
pmid = {41895443},
issn = {1083-351X},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cascade conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}

@article {pmid41896556,
year = {2026},
author = {Walsh, LH and Soni, V and Ancla, J and Somerville, V and Segata, N and Joyce, S and Sinderen, DV and Mahony, J and Shkoporov, AN and Kenny, JG and Cotter, PD and O'Sullivan, O},
title = {Mining of food metagenomes reveals an unexplored diversity of dsDNA bacteriophages.},
journal = {NPJ biofilms and microbiomes},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41522-026-00941-9},
pmid = {41896556},
issn = {2055-5008},
support = {DOMINO-101060218//European Union's Horizon Europe programme/ ; },
abstract = {Bacteriophages are key drivers of microbial ecology, co-existing and co-evolving with bacteria across diverse environments. Limitations in culturing, alongside advances in sequencing and bioinformatics, have driven the use of metagenomics to explore viral diversity. Viral-specific analysis of >3000 food metagenomes from cFMD produced the FVGC, comprising ~3400 metagenome-assembled viruses, most of which belong to novel Caudoviricetes lineages (n = 91), with only ~15% represented in IMG/VR v4. Together, these findings reveal extensive uncharacterized viral diversity in food systems. Beyond serving as a reference, the FVGC facilitates detailed investigation of virus-host interactions. Viral sequences were pervasive across microbial genomes, with several bacterial families exhibiting near-universal associations with viral elements. Bacterial antiviral defence systems were abundant and taxonomically diverse, dominated by restriction-modification systems, while CRISPR-Cas systems showed pronounced lineage-specific distributions; in contrast, viral anti-defence genes were detected at low frequency (<10% of MAVs). Host prediction linked MAVs to clinically relevant taxa, including expanded ESKAPE pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter spp., highlighting the ecological connectivity between food-associated viruses and clinically important bacteria. Antimicrobial resistance signals were scarce, suggesting minimal phage-mediated AMR dissemination in food environments. This new publicly available viral database represents a valuable resource for further exploration of viral diversity.},
}

@article {pmid41897294,
year = {2026},
author = {Zhou, Z and Zhu, L},
title = {A Computational Model for Nme1Cas9 HNH Activation Driven by Dynamic Interface Engineering at Residues S593 and W596.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897294},
issn = {2218-273X},
support = {32471296//National Natural Science Foundation of China Projects/ ; },
mesh = {Molecular Dynamics Simulation ; Thermodynamics ; Protein Engineering ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; },
abstract = {Nme1Cas9 is an encouraging genome-editing tool with high fidelity and compactness, but its applications are limited by poor catalytic efficiency compared with SpyCas9. Understanding the dynamic activation mechanism of the HNH nuclease domain is the key to breaking the kinetic bottleneck. Here, we integrated Steered Molecular Dynamics (SMD) with the Traveling-Salesman-based automated Path Searching (TAPS) algorithm to reconstruct the atomic-level activation landscape of the L1-HNH module. The simulations suggest a complex "Lifting-Rearrangement-Sliding" pathway, revealing the critical role of a "Backbone Sliding" conformation; in this step, the HNH domain rotates across the R-loop surface. A thermodynamic analysis using free energy decomposition by MM/PBSA indicates that the intrinsic instability of the wild-type HNH/R-loop interface constitutes the predominant energetic barrier. Hyperactive variants (S593Q/W596K and S593Q/W596R) can overcome this barrier by substantially increasing binding affinity to the R-loop through a "Geometry-Electrostatics Synergism": S593Q improves interfacial proximity, whereas W596K/R acts as an "Electrostatic Anchor." The results of unbiased MD simulations demonstrate that strengthened interfacial interactions effectively promote spontaneous conformational drift toward the activated state. This computational study proposes a novel in silico model for "Dynamic Interface Engineering" in which reinforcing transient interfacial contacts during conformational sliding can be an effective strategy in developing high-efficiency CRISPR-Cas effectors.},
}

Molecular Dynamics Simulation
Thermodynamics
Protein Engineering
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism

@article {pmid41864147,
year = {2026},
author = {Zhang, L and Tang, J and Feng, M and Chen, S},
title = {Development of a plasmid-free Escherichia coli strain for high-yield production of ergothioneine.},
journal = {Enzyme and microbial technology},
volume = {197},
number = {},
pages = {110850},
doi = {10.1016/j.enzmictec.2026.110850},
pmid = {41864147},
issn = {1879-0909},
mesh = {*Ergothioneine/biosynthesis ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; Plasmids/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; },
abstract = {Ergothioneine (ERG), a sulfur-containing amino acid derivative known for its antioxidant activity, has a wide range of applications in healthcare and nutrition. Escherichia coli has been extensively studied as a platform for ERG production due to its rapid growth and well-established genetic tools. However, most engineered strains rely on plasmid-based expression system, which are genetically unstable. Additionally, the requirement for antibiotics to maintain plasmid stability further limits the feasibility of plasmid-based systems for industrial-scale production. Here, we established a plasmid-free E. coli platform for ERG biosynthesis using a multi-copy chromosomal integration CRISPR-associated transposase (MUCICAT) system. We first integrated a three-gene ERG biosynthetic pathway into the E. coli genome at varying copy numbers, resulting in a five-copy strain (P5) that exhibited the highest ERG titer of 222.5 ± 5.0 mg/L. Subsequently, we reinforced the two key catalytic modules-histidine methylation and SAM biosynthesis-through iterative genomic integration of the corresponding genes, yielding a plasmid-free strain P18 that produced 370.0 ± 7.0 mg/L ERG. The engineered strain P18 exhibited excellent genetic stability, as confirmed by serial passaging. When scaled up in a 5-L bioreactor under fed-batch condition, an ERG titer of 10.1 g/L was achieved. This study demonstrates a plasmid-free ERG production strategy based on stable, multi-copy chromosomal integration of the ERG biosynthetic pathway in E. coli, highlighting its potential as an efficient platform for scalable ERG production.},
}

*Ergothioneine/biosynthesis
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
Plasmids/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems

@article {pmid41889958,
year = {2026},
author = {Ocampo, RF and Orosco, C and Huang, B and West, MS and Jain, PK and Taylor, DW},
title = {Architecture of a DNA-guided Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.19.712971},
pmid = {41889958},
issn = {2692-8205},
abstract = {CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.},
}