@article {pmid41456387,
year = {2025},
author = {Zhang, J and Liu, WJ and Ma, F and Zhang, CY},
title = {Self-priming amplification-integrated orthogonal CRISPR-Cas system for multiplexed profiling of piRNAs in clinical samples.},
journal = {Biosensors & bioelectronics},
volume = {297},
number = {},
pages = {118334},
doi = {10.1016/j.bios.2025.118334},
pmid = {41456387},
issn = {1873-4235},
abstract = {CRISPR/Cas-based biosensors hold great diagnostic potential, but they are often limited in clinical practice by insufficient sensitivity and the lack of multiplexed analysis capability. To address these issues, we develop a self-priming amplification-integrated orthogonal CRISPR-Cas (SPA-OCRISPR) system for multiplexed detection of piRNAs. This assay employs an innovative four-way junction probe that seamlessly integrates target recognition with a primer-free self-priming amplification, significantly simplifying the workflow and enhancing the ligation efficiency. High-fidelity SplitR ligase-mediated transduction guarantees the excellent specificity, efficiently eliminating the need for reverse transcription. The orthogonal trans-cleavage activities of Cas12a and Cas13a are harnessed to generate distinct fluorescent signals with minimal cross-interference for multiplexed analysis. This strategy can achieve attomolar-level sensitivity and good specificity. Moreover, it can successfully quantify breast cancer-associated piRNAs (piR-651 and piR-36026) in clinical tissues, and accurately discriminate cancerous samples from healthy ones. Importantly, this strategy exhibits good generality and it can be extended to detect colorectal cancer-related piRNAs (piR-823 and piR-54265) through simply modifying the recognition sequences of split probes, underscoring its broad potential in multiplexed profiling and clinical cancer diagnostics.},
}

@article {pmid41455873,
year = {2025},
author = {Bibi, R and George, M and Sarkar, K},
title = {RNA-guided STAT3 modification fine tunes the epigenetic and epitranscriptomic regulation of CD4 + T helper cell differentiation during non-small cell lung cancer (NSCLC).},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {2},
pages = {102},
pmid = {41455873},
issn = {1559-131X},
support = {11019/07/2018-Sch//Ministry of Tribal Affairs, Govt. of India/ ; EMDR/SG/15/2023-5901//Indian Council of Medical Research/ ; },
mesh = {Humans ; *STAT3 Transcription Factor/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/immunology/pathology ; *Lung Neoplasms/genetics/immunology/pathology ; *Epigenesis, Genetic ; Cell Differentiation/genetics ; Tumor Microenvironment/immunology ; *CD4-Positive T-Lymphocytes/immunology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Transcriptome ; },
abstract = {The accurate control of immune responses in the tumor microenvironment is crucial for augmenting anti-cancer immunity. This work examined the function of STAT3 in modulating epigenetic and epitranscriptomic pathways during the differentiation of CD4 + T helper cells in non-small cell lung cancer (NSCLC). Employing CRISPR/Cas9 genome editing, STAT3 was specifically eliminated in CD4[+]T cells derived from NSCLC patients. Functional investigations demonstrated that the reduction of STAT3 markedly enhanced the production of T helper 1 (TH1) cytokines, notably IFN-γ, while concurrently diminishing immunosuppressive signaling. Epigenetic analysis revealed significant modifications in DNA and RNA methylation patterns, along with heightened R-loop formation-alterations linked to augmented transcriptional activity of anti-tumor immune genes. Moreover, STAT3-deficient CD4[+]T cells demonstrated an enhanced ability to activate cytotoxic T lymphocytes, facilitating the targeted eradication of tumor cells. All of these effects together made the NSCLC microenvironment's immune system better at fighting cancer. Our results identify STAT3 as a crucial regulator of the genetic and epigenetic frameworks that influence T cell functionality in lung cancer. By combining RNA-guided genome editing with immune functional tests, we show that blocking STAT3 in a specific way could bring back strong anti-tumor immunity. This research underscores the therapeutic potential of STAT3-targeted therapies, presenting an innovative approach to alter T cell destiny and improve immune-mediated tumor eradication in non-small cell lung cancer (NSCLC). These methods could lead to the next generation of immunotherapies that improve clinical outcomes by fine-tuning both epigenetic and epitranscriptomic circuits.},
}

Humans
*STAT3 Transcription Factor/genetics/metabolism
*Carcinoma, Non-Small-Cell Lung/genetics/immunology/pathology
*Lung Neoplasms/genetics/immunology/pathology
*Epigenesis, Genetic
Cell Differentiation/genetics
Tumor Microenvironment/immunology
*CD4-Positive T-Lymphocytes/immunology
DNA Methylation
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Transcriptome

@article {pmid41454921,
year = {2025},
author = {Sanjay, G and Seetharam, RN and Singdevsachan, SK and Sathya, M},
title = {Microbial Systems Enhancing CAR-Based Therapies: A Synthetic Biology Paradigm for Next-Generation Cancer Immunotherapy.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {106},
pmid = {41454921},
issn = {1432-0991},
support = {Hoynoza Technologies Pvt. Ltd//Hoynoza Technologies Pvt. Ltd/ ; },
mesh = {Animals ; Humans ; *Immunotherapy/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Synthetic Biology/methods ; },
abstract = {Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Analysis of 389 peer-reviewed studies (2015-2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for:1. Synthetic antigen production and single-chain variable fragment (scFv) expression,2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation,3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70-90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads.Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.},
}

Animals
Humans
*Immunotherapy/methods
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology
*Receptors, Chimeric Antigen/genetics/immunology
*Synthetic Biology/methods

@article {pmid41454508,
year = {2025},
author = {Li, J and Wang, X and Wang, X and Qu, H and Gao, L and Zhao, Z and Luo, P and Zheng, Y},
title = {A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila.},
journal = {Polish journal of microbiology},
volume = {74},
number = {4},
pages = {484-493},
pmid = {41454508},
issn = {2544-4646},
mesh = {*Legionella pneumophila/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; Legionnaires' Disease/microbiology/diagnosis ; Water Microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Legionella pneumophila is a common environmental bacterium that can cause severe respiratory disease. In this study, a reliable, rapid, and convenient detection method for L. pneumophila was established using a combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology. First, we designed three pairs of RPA primers and two types of crRNA based on the L. pneumophila-specific mip gene. Subsequently, we optimized the primers and amplification time for the RPA reaction, the crRNA for the CRISPR/Cas12a reaction, as well as the concentration of the fluorescent probe. We successfully constructed an RPA-CRISPR/Cas12a fluorescence detection system and a portable RPA-CRISPR/Cas12a LFB. The detection systems achieved a sensitivity of 5 copies/μl and high specificity. One hundred sixty environmental water samples tested by RPA-CRISPR/Cas12a LFB showed no significant difference compared to the qPCR method, providing a reliable tool for future on-site detection.},
}

*Legionella pneumophila/isolation & purification/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Sensitivity and Specificity
Legionnaires' Disease/microbiology/diagnosis
Water Microbiology
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41453247,
year = {2025},
author = {Park, SE and Jeong, JH and Kim, YG and Park, HH},
title = {Structural analysis of predicted anti-CRISPR, ACZ01644.},
journal = {Biochemical and biophysical research communications},
volume = {797},
number = {},
pages = {153199},
doi = {10.1016/j.bbrc.2025.153199},
pmid = {41453247},
issn = {1090-2104},
abstract = {The CRISPR-Cas system provides adaptive immunity in bacteria and archaea against invading genetic elements, while anti-CRISPR (Acr) proteins have evolved in phages to counteract this defense. Here, we report the first structural and biochemical characterization of ACZ01644, a protein previously predicted to be an Acr. The crystal structure of ACZ01644 reveals a unique cone-shaped architecture composed of five α-helices and five β-strands forming a compact core, which represents a fold distinct from any known Acr family. Biochemical analyses demonstrated that ACZ01644 assembles as a trimer in solution, suggesting a potential functional relevance of this oligomeric state. However, in vitro assays revealed that ACZ01644 does not inhibit Cas9-mediated DNA cleavage, indicating that its inhibitory activity, if present, may involve other CRISPR subtypes or yet unidentified cofactors. Our findings reveal an unprecedented structural scaffold among putative Acr proteins and provide a foundation for future studies to elucidate its biological role in CRISPR-Cas regulation.},
}

@article {pmid41452514,
year = {2025},
author = {Lal, SK and Khatoon, G and Kumar, A and Kumar, K and Kumar, R and Pan, X and Kumar, S and Bhadana, VP and Pandey, A and Kumar, M and Soren, KR and Panditi, V},
title = {Genome Editing Enhanced Abiotic Stress ToleranceIn Cereal Crops.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {13},
pmid = {41452514},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Edible Grain/genetics/growth & development ; *Stress, Physiological/genetics ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Cereals are crucial sources of food for human and animal populations worldwide. Their grain and fodder primarily serve as sources of energy and nutrition. Cereal production is hampered because of the prevalent abiotic stress worldwide. Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity significantly reduce global cereal crop production. Previously, traditional breeding and transgenic technology have been promising and potent approaches used to mitigate unfavourable abiotic stresses, enhancing crop production to some extent. The recent advent of more potent genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), has revolutionized the pace of crop improvement programs. Genome-editing technology using engineered nucleases offers significant opportunities for crop improvement. Genome editing tools include Meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/CRISPR-associated protein (Cas). Among all genome-editing tools, CRISPR/Cas9 has been widely used to improve crop cultivars due to its specificity, simplicity, robustness, and flexibility. Recent progress in genome-editing technology have improved various plant traits in cereals. Among these traits, cereal genotypes have shown substantial advances in the last decade, particularly in enhanced tolerance to abiotic stress, enabled by genome-editing tools. This review summarizes the recently developed cereal cultivars for abiotic stress tolerance that employ different genome-editing technologies, including the most recent additions, prime editing and base editing. These improved cereal cultivars perform better and maintain higher yields under adverse abiotic stresses.},
}

*Gene Editing/methods
*Edible Grain/genetics/growth & development
*Stress, Physiological/genetics
CRISPR-Cas Systems
*Crops, Agricultural/genetics
*Genome, Plant
Plants, Genetically Modified/genetics

@article {pmid41430049,
year = {2025},
author = {Dong, Q and Chen, P and Guo, Z and Wei, H and Zeng, Y and Zhang, J and Men, Y and Liu, W and Sun, Y and Yang, J},
title = {Computational design of allulose-responsive biosensor toolbox for auto-inducible protein expression and CRISPRi mediated dynamic metabolic regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11562},
pmid = {41430049},
issn = {2041-1723},
support = {32271545//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biosensing Techniques/methods ; *Transcription Factors/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Escherichia coli/metabolism/genetics ; Synthetic Biology/methods ; Glucose/metabolism ; },
abstract = {Biosensors based on transcription factors (TFs) have shown extensive applications in synthetic biology. Due to the complex multi-domain structure of effector-TF-DNA, computational design of TFs remains a challenge. Here, we present the successful structure-guided computational design of the access tunnel, ligand binding, allosteric transition process for an allulose-responsive PsiR. It enables a 20-fold increase in sensitivity, reducing the EC50 of PsiR-allulose biosensors (PABs) from 16 mM to 0.8 mM, and delivers a PAB box possessing the detection range from 10 μM to 100 mM. We further validate its broader applicability in enhancing sensitivity of LacI-IPTG biosensor. Based on the developed PABs, we present the inducer-free allulose-mediated auto-inducible protein expression system, and demonstrate an allulose-triggered CRISPR interference circuit for dynamic metabolic regulation. It facilitates a 68% increase in allulose titer and achieves a high yield of 0.43 g/g glucose. This work provides the versatile TF toolbox for developing allulose-triggered regulation circuits in biotechnology application.},
}

*Biosensing Techniques/methods
*Transcription Factors/metabolism/genetics/chemistry
*CRISPR-Cas Systems
Escherichia coli/metabolism/genetics
Synthetic Biology/methods
Glucose/metabolism

@article {pmid41389205,
year = {2025},
author = {Zobel, M and Damaggio, G and Mignogna, ML and Besusso, D and Scalzo, D and Cossu, A and Trovesi, C and Crosti, M and Cortina, F and Campus, I and Formenti, G and Mazzara, S and Gregoretti, F and Antonelli, L and Oliva, G and Zuccato, C and Colonna, V and Conforti, P and Cereda, M and Rossi, RL and Maestri, S and Scolz, A and Iennaco, R and Cattaneo, E},
title = {A human CAGinSTEM platform for decoding HTT repeats' somatic instability links CAG interruption to HD pathology in neurons.},
journal = {Cell reports},
volume = {44},
number = {12},
pages = {116685},
pmid = {41389205},
issn = {2211-1247},
mesh = {Humans ; *Huntington Disease/genetics/pathology ; *Huntingtin Protein/genetics/metabolism ; *Neurons/metabolism/pathology ; *Genomic Instability ; *Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; },
abstract = {Somatic CAG instability in the mutant Huntingtin (HTT) gene is increasingly recognized as a key hallmark of Huntington's disease (HD). Using our novel human CAGinSTEM platform, we manipulated cis genetic elements influencing instability in human HD neurons, monitoring repeat length. Quality-controlled CRISPR-engineered stem cells with increasing CAG lengths and clinical haplotypes were analyzed using third-generation sequencing. Our findings link interruptions in the CAG repeat, especially the loss or duplication of the penultimate CAA of canonical alleles, to significant instability modulation. Notably, four internal CAA interruptions completely abolish CAG instability, reversing HD phenotypes such as altered striatal fate acquisition and nuclear disorganization. This platform highlights the role of cis modifiers, emphasizing the direct influence of HTT DNA repeat composition on CAG instability and providing a robust framework for modeling HTT repeat instability in vitro.},
}

Humans
*Huntington Disease/genetics/pathology
*Huntingtin Protein/genetics/metabolism
*Neurons/metabolism/pathology
*Genomic Instability
*Trinucleotide Repeats/genetics
Trinucleotide Repeat Expansion
CRISPR-Cas Systems/genetics

@article {pmid41298993,
year = {2025},
author = {Heu, CC and Benowitz, KM and Matzkin, LM and Allan, CW and LeRoy, DM and Li, X and Tabashnik, BE and Carrière, Y and Fabrick, JA},
title = {Editing the kinesin-12 gene affects responses to Bt toxin Cry1Ac in Helicoverpa zea.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {45378},
pmid = {41298993},
issn = {2045-2322},
support = {2020-33522-32268//USDA, National Institute of Food and Agriculture, Biotechnology Risk Assessment Research Grants Program/ ; 2020-67013-31924//USDA, National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; 2020-22620-023-000D//USDA, Agricultural Research Service/ ; },
mesh = {Animals ; Bacillus thuringiensis Toxins ; *Endotoxins/pharmacology ; *Gene Editing ; *Hemolysin Proteins/pharmacology ; *Kinesins/genetics ; *Moths/genetics/drug effects ; *Bacterial Proteins/pharmacology ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Bacillus thuringiensis/genetics ; *Insect Proteins/genetics ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, the evolution of resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops. Resistance to crystalline (Cry) Bt proteins including Cry1Ac produced by Bt cotton is a major problem in Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States. A previous genome-wide association study identified a nonsense point mutation in a kinesin-12 gene that was associated with resistance to Cry1Ac in a lab-selected strain of H. zea. Here, we used CRISPR/Cas9 gene editing to knock out the kinesin-12 gene in a Cry1Ac-susceptible laboratory strain, which caused a 4.0-fold increase in resistance to Cry1Ac. Conversely, gene editing that repaired the natural kinesin-12 nonsense mutation in a lab-selected resistant strain increased susceptibility to Cry1Ac by 3.8-fold. These complementary results provide compelling evidence that kinesin-12 plays a role in the mode of action of Cry1Ac against H. zea.},
}

Animals
Bacillus thuringiensis Toxins
*Endotoxins/pharmacology
*Gene Editing
*Hemolysin Proteins/pharmacology
*Kinesins/genetics
*Moths/genetics/drug effects
*Bacterial Proteins/pharmacology
*Insecticide Resistance/genetics
CRISPR-Cas Systems
Bacillus thuringiensis/genetics
*Insect Proteins/genetics

@article {pmid41298482,
year = {2025},
author = {Aguado-Alvaro, LP and Garitano, N and Esser-Skala, W and Sayers, J and Del Valle, C and Alameda, D and Mendieta-Esteban, J and Calleja-Cervantes, ME and Goñi-Salaverri, A and Zazpe, J and de Vito, AR and Marchese, F and Alignani, D and Cudini, J and Gross, T and Rábago, G and Narayan, N and Martinez, L and Martinez, S and Huntly, B and Riley, P and Gonzalez, A and Taylor-King, JP and Fortelny, N and Pelacho, B and Lara-Astiaso, D},
title = {Identification of epigenetic regulators of fibrotic transformation in cardiac fibroblasts through bulk and single-cell CRISPR screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11660},
pmid = {41298482},
issn = {2041-1723},
mesh = {Humans ; Fibrosis/genetics ; *Epigenesis, Genetic ; Single-Cell Analysis/methods ; Myofibroblasts/metabolism/pathology ; *Myocardium/pathology/metabolism/cytology ; *Fibroblasts/metabolism/pathology ; Animals ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; Mice ; Transcription Factors/metabolism/genetics ; },
abstract = {Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiates from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. In contrast, the roles of chromastin factors in myofibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors in the fibrotic transformation of primary cardiac fibroblasts. We uncover strong regulators of fibrotic states including Srcap and Kat5 chromatin remodelers. We confirm that these factors are required for functional processes underlying fibrosis including collagen synthesis and cell contractility. Using chromatin profiling in perturbed cardiac fibroblasts, we demonstrate that pro-fibrotic chromatin complexes facilitate the activity of well-characterized pro-fibrotic transcription factors. Finally, we show that KAT5 inhibition alleviates fibrotic responses in patient-derived human fibroblasts.},
}

Humans
Fibrosis/genetics
*Epigenesis, Genetic
Single-Cell Analysis/methods
Myofibroblasts/metabolism/pathology
*Myocardium/pathology/metabolism/cytology
*Fibroblasts/metabolism/pathology
Animals
Chromatin/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Cell Differentiation/genetics
Cells, Cultured
Mice
Transcription Factors/metabolism/genetics

@article {pmid41276526,
year = {2025},
author = {Murray, R and Chowdhury, MR and Botticello-Romero, NR and Desai, K and Chilakapati, SR and Chong, B and Xia, Y and Messana, A and Sobon, H and Rocha, J and Musenge, F and Camblin, A and Ciaramella, G and Sitkovsky, MV and Maldini, CR and Hatfield, SM},
title = {Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11593},
pmid = {41276526},
issn = {2041-1723},
mesh = {Animals ; Humans ; ErbB Receptors/metabolism/genetics/immunology ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; *Gene Editing/methods ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Female ; *Lung Neoplasms/therapy/immunology/genetics ; Receptor, Transforming Growth Factor-beta Type II/genetics ; *Neoplasms/therapy/immunology ; Graft vs Host Disease/prevention & control/immunology ; Mice, Inbred NOD ; },
abstract = {Chimeric Antigen Receptor (CAR) T cells have induced remarkable clinical responses in patients with hematological cancers. However, CAR T-cell therapies against solid tumors have not elicited similar outcomes since immunosuppressive barriers in the tumor microenvironment attenuate anti-tumor activity. Here, we describe a multifaceted approach to engineer allogeneic CAR T-cells resistant to both biochemical (hypoxia-adenosinergic) and immunological (PD-L1 and TGF-β) inhibitory signaling using an adenine base editor and a CRISPR-Cas12b nuclease. The resulting EGFR-targeting CAR T-cell product comprised a combination of six gene edits designed to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and overcome biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) barriers in solid tumor microenvironment of subcutaneously grown EGFR[+] human lung tumor xenografts. This combinatorial genetic disruption enhances CAR T cell effector function and anti-tumor efficacy leading to improved tumor elimination and survival in xenograft and humanized mouse solid tumor models. Our strategy confers CAR T cells resistance to multiple clinically relevant inhibitory signaling pathways that are amplified in hypoxic tumor areas and may improve the therapeutic potential of CAR T-cells against solid tumors.},
}

Animals
Humans
ErbB Receptors/metabolism/genetics/immunology
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
Mice
*Gene Editing/methods
Tumor Microenvironment/immunology
*Immunotherapy, Adoptive/methods
Xenograft Model Antitumor Assays
Cell Line, Tumor
*T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems/genetics
Female
*Lung Neoplasms/therapy/immunology/genetics
Receptor, Transforming Growth Factor-beta Type II/genetics
*Neoplasms/therapy/immunology
Graft vs Host Disease/prevention & control/immunology
Mice, Inbred NOD

@article {pmid41275110,
year = {2025},
author = {Yadav, B and Sardar, S and Yadav, A and Kumari, A and Gautam, M and Mandlik, R and Arora, S and Kumar, S and Jewaria, PK and Sonah, H and Deshmukh, R and Chinnusamy, V and Ram, H},
title = {A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1769},
pmid = {41275110},
issn = {1471-2229},
support = {BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics/growth & development ; Gene Library ; *Genes, Plant ; Gene Editing ; Gene Knockout Techniques ; Seeds/growth & development/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Edible Grain/genetics/growth & development
Gene Library
*Genes, Plant
Gene Editing
Gene Knockout Techniques
Seeds/growth & development/genetics
Plants, Genetically Modified/genetics

@article {pmid41272236,
year = {2025},
author = {Pidishetty, D and Damera, SK and Murugavel, M and Susaimanickam, PJ and Chittajallu, SNSH and Kushawah, G and Sarkar, P and Bharadwaj, SR and Mishra, R and Mariappan, I},
title = {Loss of retinal stem cell reserve and lipofuscin accumulation accelerates cone-rod degeneration and replicates Stargardt disease in abca4b null zebrafish.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44988},
pmid = {41272236},
issn = {2045-2322},
mesh = {Animals ; Zebrafish/genetics ; *Lipofuscin/metabolism ; *Stargardt Disease/genetics/pathology/metabolism ; *ATP-Binding Cassette Transporters/genetics ; Disease Models, Animal ; *Stem Cells/metabolism/pathology ; *Zebrafish Proteins/genetics/metabolism ; *Retina/pathology/metabolism ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Retinal Pigment Epithelium/metabolism/pathology ; Gene Knockout Techniques ; *Cone-Rod Dystrophies/pathology/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; *Macular Degeneration/genetics/pathology/metabolism ; },
abstract = {Mutations in ABCA4 gene causes Stargardt macular degeneration, which manifests with toxic lipofuscin deposits in the outer retina, gradual atrophy of RPE cells, followed by photoreceptor cell loss. The cone-enriched retina, with macula-like 'area-temporalis' of zebrafish are better models than rodents for studying human macular dystrophies. Here, we generated abca4b knockout zebrafish model using CRISPR/Cas9 editing and evaluated the early and late-stage retinal changes. In adult abca4b[-/-] mutants, the RPE cells exhibited hyperpigmentation, altered retinomotor behaviour and lipofuscin accumulation, but they remained viable. However, the photoreceptors underwent progressive degeneration, with a sequential loss of blue and UV cones, followed by red and green cones and finally the rod cells. This triggered the chronic activation and early depletion of retinal stem cells at the ciliary marginal zone of mutants and resulted in accelerated outer-retinal degeneration and severe visual defects, despite them retaining the Müller glia-dependant retinal repair potential.},
}

Animals
Zebrafish/genetics
*Lipofuscin/metabolism
*Stargardt Disease/genetics/pathology/metabolism
*ATP-Binding Cassette Transporters/genetics
Disease Models, Animal
*Stem Cells/metabolism/pathology
*Zebrafish Proteins/genetics/metabolism
*Retina/pathology/metabolism
Retinal Cone Photoreceptor Cells/metabolism/pathology
Retinal Rod Photoreceptor Cells/metabolism/pathology
Retinal Pigment Epithelium/metabolism/pathology
Gene Knockout Techniques
*Cone-Rod Dystrophies/pathology/metabolism/genetics
CRISPR-Cas Systems
Mutation
*Macular Degeneration/genetics/pathology/metabolism

@article {pmid35173349,
year = {2022},
author = {Shen, Y and Gomez-Blanco, J and Petassi, MT and Peters, JE and Ortega, J and Guarné, A},
title = {Structural basis for DNA targeting by the Tn7 transposon.},
journal = {Nature structural & molecular biology},
volume = {29},
number = {2},
pages = {143-151},
pmid = {35173349},
issn = {1545-9985},
support = {R01 GM129118/GM/NIGMS NIH HHS/United States ; PJT-155941//CIHR/Canada ; },
mesh = {Binding Sites/genetics ; Crystallography, X-Ray ; DNA Transposable Elements/*genetics ; DNA, Bacterial/chemistry/genetics/*metabolism ; DNA-Binding Proteins/chemistry/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Structure, Quaternary ; Substrate Specificity ; Transposases/chemistry/genetics/metabolism ; },
abstract = {Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.},
}

Binding Sites/genetics
Crystallography, X-Ray
DNA Transposable Elements/*genetics
DNA, Bacterial/chemistry/genetics/*metabolism
DNA-Binding Proteins/chemistry/genetics/metabolism
Escherichia coli/genetics/metabolism
Escherichia coli Proteins/chemistry/genetics/metabolism
Models, Molecular
Protein Interaction Domains and Motifs
Protein Structure, Quaternary
Substrate Specificity
Transposases/chemistry/genetics/metabolism

@article {pmid41451278,
year = {2025},
author = {Narra, M and Ray, A and Polley, B and Yang, H and Bhowmik, PK},
title = {AI-driven advances in plant biotechnology: sharpening the edge of plant tissue culture and genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718810},
pmid = {41451278},
issn = {1664-462X},
abstract = {The advent of artificial intelligence (AI) holds great promise for revolutionizing the fields of plant tissue culture and genome editing. Plant tissue culture is recognized as a powerful tool for rapid multiplication and crop improvement. However, the complex interactions between genetic and environmental factors generate large volumes of data, posing challenges for traditional statistical analysis methods. To address this, researchers are now employing machine learning (ML)-based and artificial neural networks (ANN) approaches to predict and optimize in vitro culture protocols thereby improving precision, sustainability, and efficiency. Integrating AI technologies such as machine learning (ML), artificial neural networks (ANN), and deep learning (DL) can significantly advance the development of data-driven models for CRISPR/Cas9 genome editing. Today, AI-driven methods are routinely applied to enhance precision in predicting on- and off-target sequence locations and editing outcomes. Additionally, predicting protein structures can provide a directed evolution framework that facilitates the creation of improved gene editing tools. However, the application of AI-based CRISPR modeling in plants is not yet fully explored. In this context, we aim to examine representative ML/DL/ANN models of CRISPR/Cas based editing employed in various organisms. This review significantly compiles a diverse set of studies and provides a clear overview of how AI is transforming the fields of plant tissue culture and genome editing. It emphasizes AI's potential to increase the efficiency and precision of biotechnological practices, making them more accessible and cost-effective. While outlining current findings, the paper sets the stage for future research, encouraging further exploration into the integration of AI with plant biotechnology.},
}

@article {pmid41450580,
year = {2025},
author = {Graves, LE and Christina, S and Mullany, KL and Alexander, IE and Falhammar, H},
title = {Exploration of the potential of genomic editing in the treatment of congenital adrenal hyperplasia.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1719376},
pmid = {41450580},
issn = {1664-2392},
mesh = {*Adrenal Hyperplasia, Congenital/genetics/therapy ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; Steroid 21-Hydroxylase/genetics ; },
abstract = {Despite life-saving glucocorticoids, therapeutic options for congenital adrenal hyperplasia (CAH) remain sub-optimal. Adrenal crisis continues to be the highest cause of mortality in individuals with CAH and even with recommended treatment regimens complications from the disease and treatments themselves persist. These patients have limited treatment options and advanced therapeutics could be a solution. Development of genetic therapies have exponentially increased in recent years. The advent of CRISPR/Cas technology has brought previously inconceivable treatment options to reality. Genomic editing could repair the defective 21-hydroxylase gene and provide a cure for 21-hydroxylase deficiency, the most common CAH variant, eliminating the current need for constant patient intervention. There are a number of technologies within reach for CAH, however, delivery of the genomic editing reagents to the elusive adrenocortical progenitor cells remains challenging. Here we discuss the complexity of CAH genetics, which has implications for choice of genomic editing strategy, and potential future strategies for the development of a cure of CAH.},
}

*Adrenal Hyperplasia, Congenital/genetics/therapy
Humans
*Gene Editing/methods
*Genetic Therapy/methods
CRISPR-Cas Systems
Animals
Steroid 21-Hydroxylase/genetics

@article {pmid41449724,
year = {2025},
author = {Plesser, E and Goldenberg, L and Kelly, G and Bdolach, E and Arad, T and Bejerano, E and Masok, O and Carmeli-Weissberg, M and Shaya, F and Sherman, A and Eyal, Y and Carmi, N},
title = {Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {6},
pages = {e70654},
pmid = {41449724},
issn = {1365-313X},
mesh = {*Flavanones/metabolism/biosynthesis ; *Citrus/genetics/metabolism ; *Gene Editing ; Plant Leaves/metabolism/genetics ; Fruit/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Taste/genetics ; Hesperidin/metabolism/analogs & derivatives ; },
abstract = {Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.},
}

*Flavanones/metabolism/biosynthesis
*Citrus/genetics/metabolism
*Gene Editing
Plant Leaves/metabolism/genetics
Fruit/genetics/metabolism
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Taste/genetics
Hesperidin/metabolism/analogs & derivatives

@article {pmid41444327,
year = {2025},
author = {Oh, D and Seok, C and Park, HW and Park, S and Lee, J and Choi, H and Jawad, A and Ham, J and Jang, H and Lee, SC and Oh, BC and Moon, C and Park, KH and Hyun, SH and Kim, D},
title = {Generation and ophthalmological characterization of oculocutaneous albinism type 1 pig models by selection-free genome editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44564},
pmid = {41444327},
issn = {2045-2322},
support = {RS-2025-00518006//National Research Foundation of Korea/ ; 2020R1A2C2101714//National Research Foundation of Korea/ ; RS-2024-00398561 , RS-2024-00399475//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries/ ; 20023068//Ministry of Agriculture, Food and Rural Affairs/ ; HR22C1363//Korea Health Industry Development Institute/Republic of Korea ; },
mesh = {Animals ; *Albinism, Oculocutaneous/genetics/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Swine ; Monophenol Monooxygenase/genetics ; CRISPR-Cas Systems ; Humans ; Electroretinography ; Phenotype ; Melanins/metabolism ; },
abstract = {Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase (TYR) gene, resulting in melanin deficiency and severe visual impairments. Although mouse models provide insights into OCA1 pathogenesis, they exhibit significant anatomical and physiological differences from humans, particularly in ocular structure and function, thereby limiting their ability to recapitulate human OCA1 phenotypes. Therefore, in this study, we generated a porcine OCA1 model by selection-free genome editing via somatic cell nuclear transfer to characterize ophthalmological features and evaluate their translational relevance to human OCA1. Our approach utilized TYR-targeting CRISPR/Cas9 ribonucleoproteins without the need for single-cell-derived clonal expansion, thus streamlining the generation process. After somatic cell nuclear transfer with TYR knockout donor cells, the embryos demonstrated normal in vitro embryonic development comparable to the control, resulting in four healthy OCA1 piglets that exhibited characteristic OCA1 phenotypes with complete melanin loss in ocular and cutaneous tissues. Comprehensive ophthalmological analyses revealed significant structural abnormalities, including marked reduction in retinal layer thickness and elevated intraocular pressure. Remarkably, electroretinography revealed selective impairment of the rod bipolar pathway with reduced b-wave amplitudes and increased oscillatory potentials, indicating disturbances in synaptic processing. Overall, our study demonstrates the efficiency and reliability of selection-free genome editing for generating porcine OCA1 models. Moreover, the ophthalmological findings provide valuable insights for exploring retinal dysfunction and pigmentation mechanisms and advancing the preclinical evaluation of potential therapeutic interventions for human OCA1.},
}

Animals
*Albinism, Oculocutaneous/genetics/pathology
*Gene Editing/methods
Disease Models, Animal
Swine
Monophenol Monooxygenase/genetics
CRISPR-Cas Systems
Humans
Electroretinography
Phenotype
Melanins/metabolism

@article {pmid41441922,
year = {2025},
author = {Govender, P and Ghai, M and Karpoormath, R},
title = {Advances in biosensors for bacterial detection and identification.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {1},
pages = {6},
pmid = {41441922},
issn = {1573-0972},
support = {PMDS2205108850//National Research Foundation/ ; },
mesh = {*Biosensing Techniques/methods ; Humans ; *Bacteria/isolation & purification/genetics/classification ; Artificial Intelligence ; Wearable Electronic Devices ; Bacterial Infections/diagnosis/microbiology ; },
abstract = {Bacterial detection and identification is paramount as it plays a key role in safeguarding human health, food safety and security. Over the past decade, biosensors have emerged as a powerful tool for bacterial detection due to their ability to provide rapid, sensitive, specific and cost-effective monitoring of bacteria. Biosensors rely on the interaction between the target analyte and biological recognition elements, which triggers a measurable signal that can be quantified, thus enabling the detection of bacteria. In recent years, nanoparticles have become a focal point in biosensor research due to their unique physical and chemical properties, enhancing their sensitivity, specificity and functionality. Artificial intelligence, microfluidics and wearable biosensor technologies are shaping the next-generation real-time bacterial monitoring tools. AI-based biosensors interpret complex biological signals and provide automated detection of bacterial pathogens. Similarly, wearable biosensors are emerging as a promising option for non-invasive detection and monitoring of wound infections. Additionally, the integration of CRISPR/Cas systems into biosensing platforms has revolutionized molecular diagnostics by enabling highly specific detection of pathogenic bacteria. In forensic sciences, biosensors are being explored for the identification of body fluids based on their unique bacterial signatures, which can assist in crime scene reconstruction and post-mortem interval estimation. Most studies that have reported on biosensors for detection of bacteria, have targeted a single analyte or bacterial species. Given the growing interest and demand for multiplexed biosensors, future research should focus on developing biosensors capable of detecting multiple bacteria simultaneously, without compromising the accuracy. Biosensors with dual functionality will be instrumental in providing an integrated solution to detect, manage and control bacterial pathogens, thereby mitigating any potential threat to human health.},
}

*Biosensing Techniques/methods
Humans
*Bacteria/isolation & purification/genetics/classification
Artificial Intelligence
Wearable Electronic Devices
Bacterial Infections/diagnosis/microbiology

@article {pmid41390487,
year = {2025},
author = {Carruthers, DN and Kinnunen, PC and Li, Y and Chen, Y and Gin, JW and Yunus, IS and Galliard, WR and Tan, S and Radivojevic, T and Adams, PD and Singh, AK and Sustarich, J and Petzold, CJ and Mukhopadhyay, A and Garcia Martin, H and Lee, TS},
title = {Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11489},
pmid = {41390487},
issn = {2041-1723},
support = {DE-AC0205CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Pseudomonas putida/metabolism/genetics ; *Machine Learning ; *Metabolic Engineering/methods ; *Hemiterpenes/metabolism ; CRISPR-Cas Systems ; Proteomics ; Metabolic Networks and Pathways/genetics ; Automation ; },
abstract = {Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway.},
}

*Pseudomonas putida/metabolism/genetics
*Machine Learning
*Metabolic Engineering/methods
*Hemiterpenes/metabolism
CRISPR-Cas Systems
Proteomics
Metabolic Networks and Pathways/genetics
Automation

@article {pmid41085147,
year = {2025},
author = {Liu, WJ and Wang, LY and Ma, F and Zhang, CY},
title = {MnO2 Nanosponge-Accelerated Cas12a Trans-Cleavage: Breaking the Kinetic Barrier for In Vivo RNA Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e11942},
doi = {10.1002/advs.202511942},
pmid = {41085147},
issn = {2198-3844},
mesh = {*Manganese Compounds/chemistry ; Humans ; *Oxides/chemistry ; *CRISPR-Cas Systems/genetics ; Kinetics ; *RNA/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a system has emerged as a promising tool for in vitro biosensing, but its in vivo applications are hindered by its inefficient intracellular delivery and suboptimal trans-cleavage kinetics. To address these challenges, a Cas12a@MnO2 nanosponge (hMNS) nanoprobe is constructed, in which hMNS as both a degradable carrier and an accelerator of CRISPR/Cas12a system for efficient imaging of RNA in living cells. The Cas12a@hMNS nanoprobe is obtained via a one-step co-assembly process. It not only facilitates synchronous cellular uptake and glutathione (GSH)-responsive release of CRISPR/Cas12a components, but also supplies adequate Mn[2+] cofactors to improve the trans-cleavage activity of Cas12a. This dual-function probe can break the kinetic barrier of conventional CRISPR/Cas12a systems due to its unique characteristics of effective cellular internalization, rapid intracellular release, and accelerated signal gain, enabling sensitive detection of mRNA down to 63.6 pM without pre-amplification. Moreover, the Cas12a@hMNS nanoprobe can profile endogenous mRNA at the single-cell level, discriminate breast cancer tissues from healthy counterparts, and real-time visualize mRNA dynamics in living cells with exceptional spatiotemporal precision. Importantly, the elongation-blocked (EB) activator-modulated CRISPR/Cas12a system can be extended to detect various intracellular biomarkers, holding promising applications in clinical diagnosis, treatment, and surveillance.},
}

*Manganese Compounds/chemistry
Humans
*Oxides/chemistry
*CRISPR-Cas Systems/genetics
Kinetics
*RNA/metabolism
*CRISPR-Associated Proteins/metabolism/genetics
Biosensing Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41058009,
year = {2025},
author = {Lu, H and Xue, C and Zhao, Y and Sun, J and Zhao, C and Zhang, X and Zhao, G and Liu, Y and Liu, H and Deng, Y and Wang, Y and Zhang, C and Liu, Y and Zeng, L and Yang, Y and Li, B and Ding, S and Zhou, L and Kuang, H and Wang, Z and Ju, W and Lin, H and Lin, J and Guo, Y and Chang, L and Zhao, H and Wang, J and Lin, J and Zheng, L and Chen, YE and Zhang, J},
title = {CRISPR-MI and scRNA-Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e12227},
doi = {10.1002/advs.202412227},
pmid = {41058009},
issn = {2198-3844},
support = {82370490//National Natural Science Foundation of China/ ; 32300958//National Natural Science Foundation of China/ ; 82200536//National Natural Science Foundation of China/ ; 2023A1515010489//Natural Science Foundation of Guangdong Province of China/ ; 20231120142637001//Shenzhen Fundamental Research Program/ ; JCYJ20230807113016034//Science and Technology project of Shenzhen of China/ ; G030410001//Medical Research Innovation Project/ ; HL109946/NH/NIH HHS/United States ; HL153710/NH/NIH HHS/United States ; //Huetwell Endowed Professorship of Cardiovascular Medicine at University of Michigan/ ; 2022JJ40812//Natural Science Foundation of Hunan Province/ ; HL109946/NH/NIH HHS/United States ; HL153710/NH/NIH HHS/United States ; },
mesh = {*Aortic Aneurysm, Abdominal/genetics/metabolism/pathology ; *Monocytes/metabolism ; Animals ; *Receptors, Immunologic/genetics/metabolism ; *Macrophages/metabolism/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice ; CRISPR-Cas Systems/genetics ; *Apoptosis/genetics ; Mice, Knockout ; Male ; Mice, Inbred C57BL ; Humans ; Disease Models, Animal ; RNA-Seq/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Gene Expression Analysis ; },
abstract = {Abdominal aortic aneurysm (AAA) is a life-threatening aortic disease without effective medication. The infiltration of monocytes into the aortic wall is critical for AAA development, but the genes and pathways regulating this process remain to be elucidated. A novel method is developed for in vivo genome-wide CRISPR/Cas9 screening of monocyte infiltration (CRISPR-MI). By combining CRISPR-MI with single-cell RNA sequencing (scRNA-Seq), this study finds that Triggering receptor expressed on myeloid cells 2 (Trem2) is a negative regulator of monocyte infiltration into the aortic wall in early AAA induction. Trem2 knockout (KO) increases the expression of adhesion molecules, chemotactic receptors, and cytokines in monocytes. Trem2 KO promotes monocyte adhesion and migration in vitro and increases monocyte infiltration into the aortic wall in vivo. However, Trem2 KO attenuates AAA development because of prominent macrophage death at the late stage. In conclusion, CRISPR-MI is a powerful tool for studying genes underlying monocyte infiltration in disease conditions in vivo. These findings reveal a dichotomous role of Trem2 in monocyte recruitment and macrophage survival during AAA.},
}

*Aortic Aneurysm, Abdominal/genetics/metabolism/pathology
*Monocytes/metabolism
Animals
*Receptors, Immunologic/genetics/metabolism
*Macrophages/metabolism/pathology
*Membrane Glycoproteins/genetics/metabolism
Mice
CRISPR-Cas Systems/genetics
*Apoptosis/genetics
Mice, Knockout
Male
Mice, Inbred C57BL
Humans
Disease Models, Animal
RNA-Seq/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Gene Expression Analysis

@article {pmid41441637,
year = {2025},
author = {Meng, Y and Chen, J and Liu, L},
title = {Functional Coupling and Evolutionary Relationships Between Toxin-Antitoxin Systems and CRISPR-Cas Systems.},
journal = {Toxins},
volume = {17},
number = {12},
pages = {},
pmid = {41441637},
issn = {2072-6651},
support = {20720250095//Fundamental Research Funds for the Central Universities/ ; 20720240046//Fundamental Research Funds for the Central Universities/ ; 32371346//National Natural Science Foundation of China/ ; 32301007//National Natural Science Foundation of China/ ; 2024J011007//Natural Science Foundation of Fujian Province/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; 2023J05008//Natural Science Foundation of Fujian Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxin-Antitoxin Systems/genetics ; *Evolution, Molecular ; *Bacteria/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *Bacterial Toxins/genetics ; },
abstract = {Bacteria encode a broad range of survival and defence systems, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, restriction-modification systems, and toxin-antitoxin (TA) systems, which are involved in bacterial regulation and immunity. The traditional view holds that CRISPR-Cas systems and TA systems are two independent defense lines in prokaryotes. However, groundbreaking studies in recent years have revealed multi-level functional coupling between them. This review systematically elaborates on this mechanism, focusing on three types of TA systems that mediate the core correlation of CRISPR-Cas systems: CreTA maintains the evolutionary stability of CRISPR-Cas systems through an addiction mechanism; CreR enables self-regulation of CRISPR-Cas expression; and CrePA provides herd immunity by triggering abortive infection after the CRISPR-Cas system has been destroyed by Anti-CRISPRS protein. Additionally, we discuss the evolutionary homology between the type III toxin AbiF and the type VI CRISPR effector Cas13, offering a new perspective for understanding the origin of CRISPR-Cas systems. These findings not only reveal the functional coupling of prokaryotic defense systems but also provide a powerful theoretical framework and practical solutions for addressing stability challenges in CRISPR technology applications.},
}

*CRISPR-Cas Systems
*Toxin-Antitoxin Systems/genetics
*Evolution, Molecular
*Bacteria/genetics/metabolism
Bacterial Proteins/genetics/metabolism
*Bacterial Toxins/genetics

@article {pmid41440302,
year = {2025},
author = {Chen, J and Liu, S and Chen, S and Mai, J and Abudukadi, M and Chen, Y and Lu, J and Li, G and Ge, C},
title = {Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440302},
issn = {2079-6374},
mesh = {*Mycoplasma hominis/isolation & purification/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques ; Humans ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.},
}

*Mycoplasma hominis/isolation & purification/genetics
*Biosensing Techniques
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques
Humans
RNA, Ribosomal, 16S/genetics

@article {pmid41349356,
year = {2025},
author = {Shahid, N and Hammond, JR},
title = {Characterization of genetically modified human embryonic kidney 293 cells lacking equilibrative nucleoside transporter subtype 2, or both subtypes 1 and 2, and the impact of their loss on sensitivity to chemotherapeutic purine/pyrimidine analogs.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {53},
number = {12},
pages = {100203},
doi = {10.1016/j.dmd.2025.100203},
pmid = {41349356},
issn = {1521-009X},
mesh = {Humans ; HEK293 Cells ; *Equilibrative Nucleoside Transporter 1/genetics/metabolism ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; CRISPR-Cas Systems ; *Purines/pharmacology/metabolism ; *Pyrimidines/pharmacology/metabolism ; *Antineoplastic Agents/pharmacology ; Biological Transport ; Gene Knockout Techniques ; },
abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.},
}

Humans
HEK293 Cells
*Equilibrative Nucleoside Transporter 1/genetics/metabolism
*Equilibrative-Nucleoside Transporter 2/genetics/metabolism
CRISPR-Cas Systems
*Purines/pharmacology/metabolism
*Pyrimidines/pharmacology/metabolism
*Antineoplastic Agents/pharmacology
Biological Transport
Gene Knockout Techniques

@article {pmid41440293,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Sotnikov, DV and Biketov, SF and Zherdev, AV and Dzantiev, BB},
title = {Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of Reporter Interactions.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440293},
issn = {2079-6374},
support = {25-16-00246//Russian Science Foundation/ ; 1.1.13//State assignment of the State Research Center of Applied Microbiology and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Kinetics ; },
abstract = {CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
Metal Nanoparticles/chemistry
Gold/chemistry
Kinetics

@article {pmid41440288,
year = {2025},
author = {Hwang, SB and Song, YJ and Park, PG},
title = {A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440288},
issn = {2079-6374},
support = {GCU-202502820001//Gachon University/ ; },
mesh = {*West Nile virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; },
abstract = {The West Nile Virus (WNV), transmitted by Culex mosquitoes as a major vector, has been reported worldwide. Also, West Nile neuroinvasive disease (WNND) caused by WNV lineage 1a and 2 neuroinvasive infections has been constantly reported with high fatality rates. Nevertheless, there are no treatments and vaccinations, so diagnosis in the early stages is important. Recently, a molecular diagnostic technique using DNA endonuclease-targeted CRISPR trans reporter (DETECTR) with the CRISPR-Cas12a system integrated with isothermal nucleic acid amplification has newly emerged. In this study, we designed a 2-Step WNV DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and sensitive WNV diagnosis. It successfully detected down to 1.0 × 10[2] RNA copies for both WNV lineage 1a and 2 with demonstrating similar sensitivity to qRT-PCR without cross-reactivity to other viruses. Additionally, we designed a 1-Step WNV DETECTR, incorporating all processing steps into a single tube, capable of detecting down to 1.0 × 10[3] RNA copies for both lineages. Furthermore, we developed a more streamlined method, the 1-Step with Filter WNV DETECTR, which achieved detection limits comparable to the 2-Step method, while reducing the processing time by 5 min. This study also explored the potential of the Punch-it™ NA-Sample Kit as an efficient alternative lysis method by comparing the detection differences across various lysis methods. Through this method, we achieved rapid and simple amplification and detection processes suitable for field diagnostics with high specificity and sufficient sensitivity. Therefore, DETECTR methods presented themselves as promising alternatives to conventional diagnostic tools, potentially overcoming financial and technical constraints in diverse medical settings.},
}

*West Nile virus/isolation & purification/genetics
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
*West Nile Fever/diagnosis/virology
Animals
Humans

@article {pmid41437670,
year = {2025},
author = {Buendia-Meraz, JA and Silva-Lucero, MD and Padilla-Mendoza, JR and Cardenas-Aguayo, MD},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's & dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e103956},
doi = {10.1002/alz70855_103956},
pmid = {41437670},
issn = {1552-5279},
mesh = {Humans ; *Alzheimer Disease/genetics/pathology/metabolism ; Cell Differentiation ; *Neural Stem Cells/metabolism/pathology ; Male ; Female ; Mutation/genetics ; Cells, Cultured ; Aged ; },
abstract = {BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder classified as either sporadic or familial (FAD), depending on the genetic component and age of onset. Understanding and applying gene-editing tools like CRISPR-Cas is of great relevance for correcting mutations and/or alterations to reverse the pathological phenotype of neurodegenerative diseases.

METHOD: To achieve this objective, we performed cell culture, immunodetection via Western blot, immunocytofluorescence, and viral transduction.

RESULT: Cell culture characterization of control and patient-derived NPCs was performed using immunocytofluorescence and Western blot detection of SOX2, Oct3/4, Nanog, Nestin associated with stem cell stages, as well as cellular maturation states (b-III-Tub, GFAP, Olig2), proliferation (Ki67), and differentiation (MAP2, NeuN). We found that FAD-derived NPCs expressed more mature markers of neuronal differentiation than control individuals' NPCs. Viral transduction with Adeno-associated viruses (AAV-9) carrying the necessary gene sequences for mutation correction in patient-derived cells was successfully performed.

CONCLUSION: Control patient cells exhibited better characterization towards stem-like phenotypes, while cells from patients with the FAD mutation of interest showed markers of cellular maturation. Viral transduction was successfully carried out; however, further analysis is needed to determine the restoration of the healthy phenotype.},
}

Humans
*Alzheimer Disease/genetics/pathology/metabolism
Cell Differentiation
*Neural Stem Cells/metabolism/pathology
Male
Female
Mutation/genetics
Cells, Cultured
Aged

@article {pmid41437291,
year = {2025},
author = {Suárez, A and Melloni, AN and Hyman, BT and Nguyen, L},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's & dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e102945},
doi = {10.1002/alz70855_102945},
pmid = {41437291},
issn = {1552-5279},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; *Caspase 8/genetics ; *Alzheimer Disease/genetics/pathology ; CRISPR-Cas Systems ; Neurons ; },
abstract = {BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. While various genetic mutations contribute to AD risk, the full genetic landscape of the disease remains unclear. Tandem repeat expansion mutations have been implicated in a subset of neurodegenerative disorders. Recently, a repeat expansion variant in CASP8 (CASP8-GGGAGA-AD-R1) has been associated with an increased risk of AD with odds ratio of 2.2 (p = 3.1 x 10[-5]). These results raise the question of how the CASP8-GGGAGA-AD-R1 sequence variant contributes to AD.

METHOD: We developed induced pluripotent stem cells (iPSC) patient derived models from AD cases with and without CASP8-GGGAGA-AD-R1 variant and control cases to study CASP8-related pathogenic pathways. We also developed CRISPR/Cas9 editing systems to excise the CASP8-GGGAGA-AD-R1 sequence to generate isogenic cell lines. Neuronal cultures developed from parental and isogenic iPSCs will be studied for disease-relevant molecular and pathogenic phenotypes.

RESULT: We have generated iPSCs using fibroblast cells derived from 4 CASP8-GGGAGA-AD-R1(+) AD, 5 CASP8-GGGAGA-C-Var(+) AD, and 5 control cases. The iPSC lines show pluripotent markers and normal karyotypes. Repeat primed PCR (RP-PCR) and long-range PCR (LR-PCR) showed the genotypes of CASP8 GGGAGA repeats are consistent with those in fibroblasts. For CRISPR/Cas9 editing of the CASP8 repeat expansion locus, we successfully cloned a plasmid to express Cas 9 protein, a fluorescence marker (mCherry and GFP) and guide RNA (gRNA) that target the upstream and downstream unique sequences of the CASP8 repeat expansion locus. Editing efficiency was tested using HEK293T cells and iPSCs, with GFP and mCherry fluorescence signals detected upon 17 and 30 hours post-transfection, demonstrating a successful expression of Cas9 proteins. LR-PCR using genomic DNA extracted from HEK293 transfected cells show expected cut size upon transfection with Cas9 systems targeting the CASP8 repeat loci.

CONCLUSION: We successfully developed patient-derived models to investigate the role of the CASP8-GGGAGA-AD-R1 repeat expansion in AD. Through CRISPR/Cas9, we demonstrated efficient excision of the mutation in HEK293 cells. Moving forward, we will apply this system to patient models to assess whether removing the mutation can mitigate disease phenotypes, providing insights into AD mechanisms and potential therapeutic strategies.},
}

Humans
*Induced Pluripotent Stem Cells
*Caspase 8/genetics
*Alzheimer Disease/genetics/pathology
CRISPR-Cas Systems
Neurons

@article {pmid41436729,
year = {2025},
author = {Wu, Y and Cai, Z and Cross, D and Noble, JR and Prest, K and Littleboy, J and Cohen, SB and Edlundh, B and Koh, JMS and Xu, R and Noor, Z and Bastami, M and Valentini, S and Richardson, L and Barthorpe, S and Arymanesh, N and Robinson, PJ and Hains, PG and Garnett, MJ and Zhong, Q and Reddel, RR and MacKenzie, KL},
title = {Large-scale drug sensitivity, gene dependency, and proteogenomic analyses of telomere maintenance mechanisms in cancer cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11337},
pmid = {41436729},
issn = {2041-1723},
support = {2017/TPG001, REG171150//Cancer Institute NSW (Cancer Institute New South Wales)/ ; CMP-01//NSW Ministry of Health (NSW Health)/ ; CMP-01//NSW Ministry of Health (NSW Health)/ ; RG24-05//Cancer Council NSW (Cancer Council New South Wales)/ ; IIRS-18-164//National Breast Cancer Foundation (NBCF)/ ; GNT1170739, H2020-SC1-DTH-2018-1//Department of Health | National Health and Medical Research Council (NHMRC)/ ; GNT1170739//Department of Health | National Health and Medical Research Council (NHMRC)/ ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/metabolism ; *Telomere Homeostasis/genetics/drug effects ; Cell Line, Tumor ; *Telomere/metabolism/genetics ; Telomerase/metabolism/genetics ; *Proteogenomics/methods ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Proteomics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Replicative immortality is a hallmark of cancer, driven by the activation of telomere maintenance mechanisms, that is yet to be therapeutically exploited. To expedite discoveries that will enable the development of therapeutics that target telomere maintenance mechanisms, this study provides a resource of telomere biology metrics for a pan-cancer panel of 976 cell lines. We generate proteomic data from data-independent-acquisition mass spectrometry for most of these cell lines and integrate pre-existing multi-omic, drug sensitivity, and molecular dependency data from CRISPR/Cas9 knock-out screens. The data illustrate a broad range and heterogeneity in telomere biology, including states that diverge from the binary model of telomere maintenance activation involving either telomerase or the Alternative Lengthening of Telomeres mechanism. Using the telomere biology metrics and multi-omic data, we derive proteomic and transcriptomic predictors of Alternative Lengthening of Telomeres and telomerase activity levels. Our investigations also reveal molecular vulnerabilities associated with the Alternative Lengthening of Telomeres mechanism and drug sensitivity correlating with telomerase activity levels. These findings illustrate opportunities for leveraging this resource to realize the potential for telomere biology-directed cancer therapeutics and companion diagnostics.},
}

Humans
*Neoplasms/genetics/drug therapy/metabolism
*Telomere Homeostasis/genetics/drug effects
Cell Line, Tumor
*Telomere/metabolism/genetics
Telomerase/metabolism/genetics
*Proteogenomics/methods
CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology
Proteomics
Drug Resistance, Neoplasm/genetics

@article {pmid41434963,
year = {2025},
author = {Ramos, DM and Nelson, MP and Calzada, L and Krishna, S and Neuman, SS and Weller, C and Johnson, N and Nalls, MA and Lara, E and Qi, A and Santiana, M and Pantazis, C and Skarnes, WC and Singleton, A and Cookson, MR and Ward, M},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's & dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e097245},
doi = {10.1002/alz70855_097245},
pmid = {41434963},
issn = {1552-5279},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neurons/metabolism ; *CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics ; Gene Knockdown Techniques ; },
abstract = {BACKGROUND: The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever induced pluripotent stem cell (iPSC) genome engineering project, modeling over 100 ADRD mutations in high-quality isogenic human iPSCs. iNDI leverages unbiased CRISPRi screens as a powerful tool to identify fundamental mechanisms and modifiers of disease. However, current CRISPRi molecular tools are poorly optimized for use in iPSC-derived neurons (iNeurons). Here we develop a Cre-lox inducible CRISPRi system (CRISPRi-Cre), enabling gene knockdown upon Cre delivery to postmitotic iNeurons, and identification of neuron-specific, disease-relevant modifiers.

METHOD: We modified a plasmid carrying a potent Zim3-dCas9 transcriptional repressor to include a strong floxed STOP cassette upstream of the Zim3 start codon. We leveraged HaloTag-TDP43 and HaloTag-FUS iSPCs from the iNDI project paired with flow cytometry to validate leakiness and responsiveness to Cre in iPSCs and iNeurons treated with sgRNAs. We then performed a genome-wide CRISPRi survival screen in iNeurons to demonstrate broad functionality of this inducible CRISPRi system with over 20,000 sgRNAs. Finally, we use CRISPRi-Cre to identify neuron-specific regulators of neuronal activity in iNeurons.

RESULT: We demonstrate that in the absence of Cre, dCas9 is inactive. Delivery of lentivirus-Cre to iNeurons activates dCas9, resulting in potent gene knockdown. In genome-wide CRISPRi screens, we show that CRISPRi-Cre identifies many of the same hits observed in screens using constitutive-active dCas9, and importantly uncovers novel neuron-specific hits not identified in previous CRISPRi screens.

CONCLUSION: Here, we developed a robust Cre-inducible CRISPRi system that enables post-mitotic gene knockdown in iPSC-derived neurons. Our CRISPRi screens identify neuron-specific hits, demonstrating the utility of our tool to help uncover disease-relevant mechanisms, modifiers, and potential therapeutic targets in relevant cell types.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Neurons/metabolism
*CRISPR-Cas Systems
*Neurodegenerative Diseases/genetics
Gene Knockdown Techniques

@article {pmid41432353,
year = {2025},
author = {Tang, X and Ju, D and Hu, H},
title = {A Dual CRISPR-Cas/Cre-loxP Genome Engineering Strategy for Stable Uricase Expression in Food-Grade Probiotics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00774},
pmid = {41432353},
issn = {2161-5063},
abstract = {The development of robust, food-grade microbial chassis with tailored metabolic functions is critical for advancing synthetic biology applications in health and nutrition. Here, we report a dual genome engineering strategy that integrates CRISPR-Cas9-mediated knock-in with Cre/loxP-driven genome reduction to streamline the genome of Lactococcus lactis NZ9000 and enable stable expression of a high-activity uricase variant. The resulting strain, NZ9000::UA[T]-ΔD6, demonstrated enhanced enzymatic performance in vitro, achieving 2.34 U/mL activity and complete degradation of ∼500 μM urate within 20 h. Beyond improved catalytic output, this dual-system approach established a genetically stable and biosafe probiotic chassis with moderate colonization capacity in the murine gut. The integration of CRISPR-Cas and Cre/loxP techniques in this work is intended to enhance the expression of heterologous genes in the chassis strain, while providing a versatile platform for the rational design of food-grade probiotics and offering a general strategy for constructing living biotherapeutic agents with targeted metabolic activities.},
}

@article {pmid41432281,
year = {2025},
author = {Liu, XL and Liu, L and Cheng, L},
title = {Sequence-independent optical regulation of CRISPR/Cas editing using star-shaped crRNA dendrimers.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc06011g},
pmid = {41432281},
issn = {1364-548X},
abstract = {Precise spatiotemporal control of CRISPR/Cas editing is vital for studying dynamic processes and ensuring therapeutic safety. We present a single-site photolabile crRNA dendrimer platform enabling robust, sequence-independent optical regulation of Cas9 and Cas12a. This simple, universal strategy achieves rapid OFF-to-ON control with minimal leakage, advancing programmable, light-responsive genome editing for biomedical applications.},
}

@article {pmid41417859,
year = {2025},
author = {Bradford, J and Joy, D and Winsen, M and Meurant, N and Wilkins, M and Wilson, LOW and Bauer, DC and Perrin, D},
title = {Democratising high performance computing for bioinformatics through serverless cloud computing: A case study on CRISPR-Cas9 guide RNA design with Crackling Cloud.},
journal = {PLoS computational biology},
volume = {21},
number = {12},
pages = {e1013819},
doi = {10.1371/journal.pcbi.1013819},
pmid = {41417859},
issn = {1553-7358},
mesh = {*Cloud Computing ; *Computational Biology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Software ; },
abstract = {Organisations are challenged when meeting the computational requirements of large-scale bioinformatics analyses using their own resources. Cloud computing has democratised large-scale resources, and to reduce the barriers of working with large-scale compute, leading cloud vendors offer serverless computing, a low-maintenance and low-cost model that provides ample resources for highly scalable software applications. While serverless computing has broad use, its adoption in bioinformatics remains poor. Here, we demonstrate the most extensive use of high-performance serverless computing for bioinformatics by applying the available technologies to CRISPR-Cas9 guide RNA (gRNA) design. Our adaptation of the established gRNA design tool, named Crackling, implements a novel, cloud-native and serverless-based, high-performance computing environment using technologies made available by Amazon Web Services (AWS). The architecture, compatible with technologies from all leading cloud vendors, and the AWS implementation, contributes to an effort of reducing the barrier to large computational capacity in bioinformatics and for CRISPR-Cas9 gRNA design. Crackling Cloud can be deployed to any AWS account, and is freely available on GitHub under the BSD 3-clause license: https://github.com/bmds-lab/Crackling-AWS.},
}

*Cloud Computing
*Computational Biology/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
Software

@article {pmid41284608,
year = {2025},
author = {Zhu, W and Qi, T and Wu, Z and Zhong, W and Yan, L and Feng, J and Jin, F and Chen, W and Cai, Z and Rui, Y and Maria Da Costa, E and Liu, Q and Fu, Q and Zheng, L},
title = {One-Pot Photocontrolled CRISPR-Cas12b Coupled with Loop-Mediated Isothermal Amplification Assay for Point-of-Care Test of Mycoplasma pneumonia.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9323-9334},
doi = {10.1021/acssensors.5c01679},
pmid = {41284608},
issn = {2379-3694},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Mycoplasma pneumoniae/isolation & purification/genetics ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Point-of-Care Testing ; Humans ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; DNA, Bacterial/genetics/analysis ; },
abstract = {Mycoplasma pneumoniae (MP) is a highly prevalent respiratory pathogen, making the development of point-of-care testing (POCT) methods for its detection essential. The integration of loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b systems demonstrates remarkable specificity and offers promising potential for MP POCT application. However, the current one-pot LAMP/CRISPR-Cas12b system (HOLMESv2) faces the challenge of low sensitivity due to the premature cleavage of the template by CRISPR, which limits its practical utility. To address this, this study introduces a photocontrolled HOLMESv2 (pHOLMESv2) assay using gRNA with an NPOM-modified spacer region. This modification prevents full base pairing between the gRNA and MP DNA, thereby keeping the CRISPR-Cas12b system in an inactive condition during the LAMP reaction and avoiding unintended cleavage of the DNA template. After completion of the LAMP reaction, light irradiation eliminates the NPOM group from the gRNA, restoring its activity to cleave the LAMP products, resulting in a fluorescence signal. The pHOLMESv2 assay successfully addresses the issue of premature DNA template cleavage, improving the limit of detection (LoD) by 133-fold (7.5 copies/μL). Additionally, this method enables direct detection of samples treated with nucleic acid release agents, eliminating the need for complex extraction, and features lyophilized reagents for enhanced stability, storage, and transport. The efficacy of pHOLMESv2 was assessed with 160 clinical MP samples, achieving a sensitivity of 99.0% and a specificity of 100.0%. The pHOLMESv2 assay, when combined with the developed smartphone-based amplification reader, provides a highly sensitive, specific, portable, and cost-effective MP detection, indicating its potential for significant diagnostic use.},
}

*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
*Mycoplasma pneumoniae/isolation & purification/genetics
*Pneumonia, Mycoplasma/diagnosis/microbiology
*Point-of-Care Testing
Humans
*Molecular Diagnostic Techniques/methods
Limit of Detection
DNA, Bacterial/genetics/analysis

@article {pmid41218722,
year = {2026},
author = {Madugula, SS and Jayasinghe-Arachchige, VM and Norgan Radler, CR and Wang, S and Liu, J},
title = {Structure-Based Classification of CRISPR/Cas9 Proteins: A Machine Learning Approach to Elucidating Cas9 Allostery.},
journal = {Journal of molecular biology},
volume = {438},
number = {2},
pages = {169538},
doi = {10.1016/j.jmb.2025.169538},
pmid = {41218722},
issn = {1089-8638},
mesh = {*Machine Learning ; Allosteric Regulation ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Molecular Dynamics Simulation ; Streptococcus pyogenes/genetics/enzymology ; Gene Editing/methods ; Protein Conformation ; },
abstract = {The CRISPR/Cas9 system is a powerful gene-editing tool. Its specificity and stability rely on complex allosteric regulation. Understanding these allosteric regulations is essential for developing high-fidelity Cas9 variants with reduced off-target effects. Here, we used a novel structure-based machine learning (ML) approach to systematically identify long-range allosteric networks in Cas9. Our ML model was trained using all available Cas9 structures, ensuring a comprehensive representation of Cas9's structural landscape. We then applied this model to Streptococcus pyogenes Cas9 (SpCas9) to demonstrate the feature selection process. Using Cα-Cα inter-residue distances, we mapped key allosteric networks and refined them through a two-stage SHAP feature selection (FS) strategy, reducing a vast feature space to 28 critical Lysine-Arginine (Lys-Arg) residue pairs that mediate SpCas9 interdomain communication, stability, and specificity. These Lys-Arg pairs initially shared a 46.5 Å inter-residue distance, but molecular dynamics simulations revealed distinct stabilization behaviors, indicating a hierarchical allosteric network. Further mutational analysis of R78A-K855A (M1) and R765A-K1246A (M2) identified an "electrostatic valley," a stabilizing network where positively charged residues interact with negatively charged DNA to maintain SpCas9's structural integrity. Disrupting this valley through direct (M2) or allosteric (M1) mutations destabilized SpCas9's DNA-bound conformation, leading to distinct pathways for improving SpCas9 specificity. This study provides a new framework for understanding allostery in Cas9, integrating ML-driven structural analysis with MD simulations. By identifying key allosteric residues and introducing the electrostatic valley as a central concept, we offer a rational strategy for engineering high-fidelity Cas9 variants. Beyond Cas9, our approach can be applied to uncover allosteric hotspots in other enzyme regulations and rational protein design.},
}

*Machine Learning
Allosteric Regulation
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
Molecular Dynamics Simulation
Streptococcus pyogenes/genetics/enzymology
Gene Editing/methods
Protein Conformation

@article {pmid41037798,
year = {2025},
author = {Park, HJ and Kim, J and Choi, J and Ryou, C and Shin, E and Lee, JY},
title = {Targeted genome editing of ZKSCAN3 mitigates the neurotoxicity caused by mutant HTT (huntingtin) in a Huntington disease animal model and three-dimensional cell culture of Huntington disease.},
journal = {Autophagy},
volume = {21},
number = {12},
pages = {3398-3412},
doi = {10.1080/15548627.2025.2569965},
pmid = {41037798},
issn = {1554-8635},
mesh = {Animals ; *Huntington Disease/genetics/pathology ; Humans ; Disease Models, Animal ; Induced Pluripotent Stem Cells/metabolism ; Autophagy/genetics ; *Huntingtin Protein/genetics/metabolism/toxicity ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Spheroids, Cellular/metabolism ; Mice ; *Mutation/genetics ; Lysosomes/metabolism ; Neurons/metabolism/pathology ; },
abstract = {Huntington disease (HD) is a neurodegenerative disease caused by the expression of a mutant form of HTT (huntingtin; mHTT), caused by an abnormal expansion of polyglutamine in HTT. In HD, macroautophagy/autophagy dysfunction can cause mHTT accumulation. Moreover, the promotion of autophagy is considered a therapeutic strategy for the treatment of HD. ZKSCAN3 (zinc finger with KRAB And SCAN domains 3) has been identified as a transcriptional repressor of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal functions. In this study, we conducted CRISPR-Cas9-based gene ablation to disrupt ZKSCAN3 in HD animal models and HD patient-induced pluripotent stem cell (iPSC) -derived three-dimensional (3D) spheroids. In animal models of HD, targeted in vivo zkscan3 ablation via a single adeno-associated virus (AAV) mediated CRISPR-Cas9 approach resulted in reduced mHTT levels, leading to improvements in both behavioral symptoms and the brain environment. Furthermore, CRISPR-Cas9 mediated ablation of ZKSCAN3 in 3D spheroids from HD patient-derived iPSC resulted in increased autophagy and lysosomal function, along with reduced mHTT accumulation. Specifically, in iPSC-derived neurons from HD patients, ZKSCAN3-depleted neurons demonstrated increased lysosomal function and reduced oxidative stress compared to controls. Additionally, transcriptional analysis of ZKSCAN3-edited neurons revealed an increased expression of genes involved in synaptic function and transporter activity. Taken together, these results suggest that in HD treatment strategies for improving neuronal function and the brain environment, ZKSCAN3 downregulation in neurons by autophagy activation may improve the brain environment through neuronal self-repair.Abbreviations: 2D: two-dimensional; 3D: three-dimensional; 4-HNE: 4-hydroxynonenal; AAV: adeno-associated virus; AD: Alzheimer disease; Aβ: beta-amyloid; DAPI: 4,6-diamidino-2-phenylindole; GFP: green fluorescent protein; HD: Huntington disease; HTT: huntingtin; IXMC: ImageXpress microconfocal high-content imaging system; Indel: insertion or deletion; iPSC: induced pluripotent stem cell; LAMP1: lysosomal-associated membrane protein 1; mHTT: mutant huntingtin; NPCs: neural precursor cells; RBFOX3/NeuN: RNA binding fox-1 homolog 3; PD: Parkinson disease; RNP: ribonucleoprotein; sgRNAs: single guide RNAs; ST: striatum; TFEB: transcription factor EB; TUBB3/Tuj-1: tubulin beta 3 class III; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.},
}

Animals
*Huntington Disease/genetics/pathology
Humans
Disease Models, Animal
Induced Pluripotent Stem Cells/metabolism
Autophagy/genetics
*Huntingtin Protein/genetics/metabolism/toxicity
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Transcription Factors/genetics/metabolism
Spheroids, Cellular/metabolism
Mice
*Mutation/genetics
Lysosomes/metabolism
Neurons/metabolism/pathology

@article {pmid41390513,
year = {2025},
author = {Vermeulen, M and Craig, AW and Babak, T},
title = {Challenges and opportunities for oncology drug repurposing informed by synthetic lethality.},
journal = {NPJ systems biology and applications},
volume = {11},
number = {1},
pages = {143},
pmid = {41390513},
issn = {2056-7189},
support = {PJT 178214//Canadian Institutes of Health Research Project Grant/ ; },
mesh = {Humans ; *Drug Repositioning/methods ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/genetics/drug therapy ; Gene Knockout Techniques ; Mutation ; },
abstract = {Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A-mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models.},
}

Humans
*Drug Repositioning/methods
*Synthetic Lethal Mutations/genetics
Cell Line, Tumor
CRISPR-Cas Systems/genetics
*Antineoplastic Agents/pharmacology/therapeutic use
*Neoplasms/genetics/drug therapy
Gene Knockout Techniques
Mutation

@article {pmid41205600,
year = {2025},
author = {Saini, M and Castro-Giner, F and Hotz, A and Sznurkowska, MK and Nüesch, M and Cuenot, FM and Budinjas, S and Bilfeld, G and Ildız, ES and Strittmatter, K and Krol, I and Diamantopoulou, Z and Paasinen-Sohns, A and Waldmeier, M and Cássio, R and Kreutzer, S and Kontarakis, Z and Gvozdenovic, A and Aceto, N},
title = {StealTHY: An immunogen-free CRISPR platform to expose concealed metastasis regulators in immunocompetent models.},
journal = {Cell},
volume = {188},
number = {26},
pages = {7591-7609.e32},
doi = {10.1016/j.cell.2025.10.007},
pmid = {41205600},
issn = {1097-4172},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Humans ; *Neoplasm Metastasis/genetics ; Mice ; Cell Line, Tumor ; Immunocompetence ; *Neoplasms/genetics/immunology/pathology ; Mice, Inbred C57BL ; Female ; },
abstract = {CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to broad immunogenicity of bacterial nucleases and reporters. Here, we show how conventional CRISPR screens in tumor grafts are systematically jeopardized by immunoediting in syngeneic and humanized immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification. To resolve this, we present StealTHY, an immunogen-free CRISPR platform compatible with virtually all immunocompetent designs, enabling preservation of clonal architecture and exposing previously concealed cancer vulnerabilities. Among these, we identify the AMH-AMHR2 axis as a formerly unappreciated metastasis target. Thus, with StealTHY, we provide a new resource to expand the applicability of CRISPR screens to immunocompetent models, including humanized tumor grafts, revealing metastasis regulators of therapeutic relevance.},
}

Animals
*CRISPR-Cas Systems/genetics
Humans
*Neoplasm Metastasis/genetics
Mice
Cell Line, Tumor
Immunocompetence
*Neoplasms/genetics/immunology/pathology
Mice, Inbred C57BL
Female

@article {pmid41430372,
year = {2025},
author = {Kim, GE and Lee, SY and Kang, YJ and Bin Jin, H and Park, HH},
title = {AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages.},
journal = {Communications biology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s42003-025-09417-6},
pmid = {41430372},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems, providing valuable tools for regulating genome editing. Here, we present the crystal structure of AcrIIA19, a plasmid-encoded Type II-A CRISPR-Cas system inhibitor that targets Cas9. AcrIIA19 adopts a previously uncharacterized fold and forms a stable homodimer. Biochemical assays revealed that AcrIIA19 binds selectively to the wedge (WED) domain of Cas9, a conserved structural interface critical for single guide RNA-DNA duplex stabilization and catalysis. This interaction disrupts Cas9 activity at multiple stages, independent of the order of complex assembly. Notably, AcrIIA19 exhibits broad-spectrum inhibition across divergent Cas9 orthologs, including Streptococcus pyogenes and Staphylococcus aureus Cas9, by exploiting a conserved WED domain vulnerability. Our findings establish AcrIIA19 as a versatile Cas9 inhibitor and highlight the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools in biotechnology and therapeutic applications.},
}

@article {pmid41428734,
year = {2025},
author = {Zhu, C and Xiao, D and Wang, Y and Han, H and Qin, C and Liu, S and Chen, X and Xiao, H and Chen, X and Shi, J and Tang, J and Shen, J and Song, H},
title = {Molecular basis of NFIB-mediated regulation of oncogenic transcription.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428734},
issn = {1362-4962},
support = {Z2023033//Hunan Health Commission Key Clinical Specialty Major Research Project/ ; NFPS-JJ-501348//Chinese National Key Clinical Specialty Program/ ; 82272508//National Natural Science Foundation of China/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; 0007/2022/AKP, 0068/2023/ITP2, 0143/2025/ITP2//Macau Science and Technology Development Fund/ ; MYRG-GRG2024-00283-ICMS-UMDF, SRG2023-00054-ICMS//University of Macau/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; },
mesh = {Humans ; *NFI Transcription Factors/genetics/chemistry/metabolism ; *Gene Expression Regulation, Neoplastic ; DNA/metabolism/chemistry/genetics ; Cell Line, Tumor ; *Transcription, Genetic ; CRISPR-Cas Systems ; Protein Binding ; *Neoplasms/genetics/pathology/metabolism ; Cell Proliferation/genetics ; Oncogenes ; Models, Molecular ; Cell Movement/genetics ; Transcriptional Activation ; },
abstract = {The Nuclear Factor I (NFI) family of transcription factors orchestrates key regulatory programs in development, differentiation, and metabolism, with dysregulation implicated in diverse pathological conditions, including cancer. Among the paralogs, NFIB has emerged as an oncogenic driver in multiple tumor types, yet the mechanisms through which it engages DNA and directs oncogenic transcriptional programs remain undefined. Here, using cancer cells with high NFIB expression, we demonstrate that NFIB promotes malignant phenotypes, as CRISPR-Cas9 knockout impairs proliferation, migration, and invasion. Transcriptomic profiling reveals that NFIB regulates a cancer-enriched gene network that includes FGFR3 and PDGFRB. Biophysical analyses show that NFIB, including its DNA-binding domain, functions as a monomer and binds DNA with strict 1:1 stoichiometry. High-resolution crystal structures of NFIB DNA-binding domain bound to ChIP-seq-derived DNA motifs reveal a monomeric binding mode mediated by conserved base-specific interactions with the TGGCA sequence, providing an atomic view of NFIB-DNA recognition. Mutational disruption of key DNA-contacting residues abolishes DNA binding and transcriptional activation, linking atomic-level recognition to oncogenic transcriptional regulation. Together, these findings elucidate the structural mechanism underlying NFIB function in cancer and establish a framework for therapeutic strategies targeting NFIB-driven malignancies.},
}

Humans
*NFI Transcription Factors/genetics/chemistry/metabolism
*Gene Expression Regulation, Neoplastic
DNA/metabolism/chemistry/genetics
Cell Line, Tumor
*Transcription, Genetic
CRISPR-Cas Systems
Protein Binding
*Neoplasms/genetics/pathology/metabolism
Cell Proliferation/genetics
Oncogenes
Models, Molecular
Cell Movement/genetics
Transcriptional Activation

@article {pmid41428733,
year = {2025},
author = {Yang, Z and Yu, M and Li, P and Li, Z and Teng, Y and Zhou, Y and Zhao, M and Liu, C and Zhao, Z and Wang, Z and Li, J and Jing, Y and Li, Y and Zhao, H and Song, W and Bian, C and Zhao, H and Chen, J and Xin, B and Lai, J},
title = {Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428733},
issn = {1362-4962},
support = {//Agriculture Science and Technology/ ; 2023YFD1202900//National Key Research and Development Program of China/ ; PC2023A01004//Pinduoduo-China Agricultural University/ ; //Agriculture Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Evolution, Molecular ; Oryza/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) adaptive immune systems provide sequence-specific mechanisms for targeting foreign DNA or RNA and have been widely used in genome editing and DNA detection. Type V CRISPR-Cas systems are characterized by a single RNA-guided RuvC domain-containing effector, Cas12. Here, through comprehensive mining of large-scale genomic and metagenomic data from microbial sources, we identified a new Class 2 CRISPR-Cas effector superfamily, designated Casδ, comprising three members with protein sizes ranging from 867 to 936 amino acids. Biochemical analyses revealed that Casδ-1 functions as a single RNA-guided endonuclease with specific recognition of 5'-RYR-3' protospacer-adjacent motifs, where R represents A or G, and Y represents T or C. Casδ-1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity. Casδ-1 mediates efficient genome editing across species, achieving up to 60% indel rates in human cells while generating homozygous knockout lines in two agriculturally important monocot species (Oryza sativa and Zea mays) through stable transformation. Structural and evolutionary analyses reveal Casδ as an evolutionary transitional nuclease bridging Cas12n and canonical type V systems, featuring a C-terminal loop that is essential for activity. Collectively, Casδ is an evolutionarily distinct, compact (<1000 aa), tracrRNA-free CRISPR system enabling versatile cross-kingdom genome editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
Animals
*CRISPR-Associated Proteins/genetics/metabolism/chemistry
Genome, Plant
RNA, Guide, CRISPR-Cas Systems/genetics
Zea mays/genetics
Evolution, Molecular
Oryza/genetics

@article {pmid41428728,
year = {2025},
author = {Gervais, NC and Rogers, RKJ and Robin, MR and Shapiro, RS},
title = {HyperdCas12a-based multiplexed genetic regulation in Candida albicans.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428728},
issn = {1362-4962},
support = {RGPIN-2018-4914//Natural Sciences and Engineering Research Council of Canada/ ; //NSERC/ ; //Canada Research Chair/ ; RGPIN-2018-4914//NSERC/ ; },
mesh = {*Candida albicans/genetics/metabolism/drug effects ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Ergosterol/biosynthesis ; Fungal Proteins/genetics/metabolism ; Drug Resistance, Fungal/genetics ; },
abstract = {Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPR activation and CRISPR interference tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.},
}

*Candida albicans/genetics/metabolism/drug effects
*CRISPR-Cas Systems
*Gene Expression Regulation, Fungal
Ergosterol/biosynthesis
Fungal Proteins/genetics/metabolism
Drug Resistance, Fungal/genetics

@article {pmid41428487,
year = {2025},
author = {Deng, C and Hu, J and Chen, Q and Zhou, S and Ni, J},
title = {Expanded global groundwater microbial diversity reveals bioprospecting potential.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116760},
doi = {10.1016/j.celrep.2025.116760},
pmid = {41428487},
issn = {2211-1247},
abstract = {Although the terrestrial subsurface harbors a substantial fraction of Earth's microbial biomass, the genomic diversity of groundwater microbiomes and their potential for bioprospecting remain poorly characterized. Here, we recovered 44,320 bacterial and archaeal genomes from in-house and publicly available metagenomic datasets, establishing a large-scale groundwater microbiota catalog (GWMC) spanning 167 phyla, including four candidate phyla and over 12,000 previously uncharacterized species. This unprecedented phylogenetic diversity was accompanied by a bimodal genome size distribution (0.3-12.8 Mbp), revealing divergent strategies of genomic allocation. By mining extensive genomic resources, we found that small genomes prioritized molecular defense and redox regulation, whereas large genomes frequently harbored greater biosynthetic potential. Notably, we establish the largest selenoprotein catalog to date and highlight groundwater as an overlooked hotspot of microbial selenium metabolism. Overall, this work advances our understanding of microbial diversity in aquifers and uncovers underexplored genomic resources with potential for biotechnology and biomedicine.},
}

@article {pmid41428463,
year = {2025},
author = {Ye, T and Xue, M and Chen, H and Yue, S and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Allosteric Aptamer CRISPR/Cas Activation Enables Non-competitive ATP Detection and Meat Freshness Assessment.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c12150},
pmid = {41428463},
issn = {1520-5118},
abstract = {CRISPR/Cas-based aptasensors (Cas-aptasensors) hold great promise for detecting non-nucleic acid targets, yet their intrinsic competitive recognition mechanism imposes a trade-off between transduction efficiency and background leakage. In this study, we developed a Cas-aptasensor that employs a non-competitive recognition mechanism. In our design, the aptamer-target interaction accelerates the toehold-mediated strand displacement reaction and exposes a second toehold domain. The CRISPR/Cas system is ultimately activated via a cascade strand-displacement reaction, which is hindered in the absence of the target and, thus, delays activation. We demonstrated the applicability of this non-competitive Cas-aptasensor for the detection of ATP, achieving a detection limit as low as 1.0 nM within 45 min. Furthermore, we successfully applied this method to ATP detection in complex matrices and to assess the freshness of diverse meat products across different storage temperatures. Overall, this work advances the design of Cas-aptasensors and expands their potential applications in food safety monitoring.},
}

@article {pmid41425600,
year = {2025},
author = {Fazeli, A and Ullrich, E and Cathomen, T and Bexte, T},
title = {Engineering with care: safety assessment platforms for CRISPR-modified natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1711414},
pmid = {41425600},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Immunotherapy, Adoptive/methods/adverse effects ; },
abstract = {CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical in vitro assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.},
}

*Killer Cells, Natural/immunology/metabolism/transplantation
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Immunotherapy, Adoptive/methods/adverse effects

@article {pmid41424917,
year = {2025},
author = {Sharma, S and Saroha, NK and Sehrawat, A and Tang, G and Singh, D and Teotia, S},
title = {Emerging tools in plant genome editing.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1588089},
pmid = {41424917},
issn = {2673-3439},
abstract = {Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.},
}

@article {pmid41390669,
year = {2025},
author = {Teske, M and Wertheimer, T and Butz, S and Zwicky, P and Mallona, I and Nopper, SL and Münz, C and Elling, U and Lancrin, C and Becher, B and Grosso, AR and Baubec, T and Schmolka, N},
title = {Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11412},
pmid = {41390669},
issn = {2041-1723},
support = {186012//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; FAN//Universität Zürich (University of Zurich)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Mesoderm/cytology/metabolism ; Cell Differentiation/genetics ; Cell Lineage/genetics ; *Hematopoietic Stem Cells/cytology/metabolism ; Gene Expression Regulation, Developmental ; Mouse Embryonic Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; Hematopoiesis/genetics ; },
abstract = {During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm forming haematopoietic progenitors. This study aims to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employ a murine embryonic stem cell model that recapitulates embryonic blood development, and perform targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators. We identify the transcription factors ETV2, LDB1, SMAD1, SIX4 and ZBTB7b as regulators of haemato-endothelial mesoderm commitment. Embryonic stem cells lacking these regulators give rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovers the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
*Transcription Factors/genetics/metabolism
Mesoderm/cytology/metabolism
Cell Differentiation/genetics
Cell Lineage/genetics
*Hematopoietic Stem Cells/cytology/metabolism
Gene Expression Regulation, Developmental
Mouse Embryonic Stem Cells/cytology/metabolism
*Endothelial Cells/cytology/metabolism
Hematopoiesis/genetics

@article {pmid41387334,
year = {2025},
author = {Song, N and Tian, G and Li, H and Zhang, L and Wang, Y and Zhao, W and Yao, C and Yang, D},
title = {DNA Nanoflowers Efficiently Encapsulate Photodynamic Agents and CRISPR/Cas9 for Synergistic Pancreatic Cancer Therapy.},
journal = {Nano letters},
volume = {25},
number = {51},
pages = {17693-17701},
doi = {10.1021/acs.nanolett.5c04676},
pmid = {41387334},
issn = {1530-6992},
mesh = {*Pancreatic Neoplasms/drug therapy/pathology/genetics/therapy/metabolism ; *Photochemotherapy/methods ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Cell Line, Tumor ; *Photosensitizing Agents/chemistry/pharmacology/administration & dosage/therapeutic use ; Chlorophyllides ; *DNA/chemistry ; Reactive Oxygen Species/metabolism ; Porphyrins/chemistry/pharmacology/administration & dosage ; Hemin/chemistry ; Apoptosis/drug effects ; Gene Editing ; Ribonucleoproteins/genetics ; G-Quadruplexes ; },
abstract = {Photodynamic therapy (PDT) holds significant promise for treating pancreatic cancer by utilizing photosensitizers to generate reactive oxygen species (ROS) that induce tumor cell death. However, the therapeutic efficacy of PDT is hindered by inadequate ROS accumulation. Herein, we develop a DNA nanoflower that enables the controlled codelivery of Cas9 ribonucleoprotein (RNP), hemin, and chlorin e6 for synergistic PDT. The Cas9 RNP selectively knocks out the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), thereby increasing cancer cells' sensitivity to ROS. Simultaneously, the G-quadruplex/hemin complex catalyzes the conversion of endogenous H2O2 into O2, alleviating tumor hypoxia and supplying additional oxygen for PDT. This synergistic approach substantially amplifies ROS accumulation by attenuating ROS elimination and enhancing ROS generation, demonstrating high gene editing efficiency, significant Nrf2 down-regulation, elevated apoptosis, and remarkable antitumor efficacy in pancreatic cancer cells and a mouse model, underscoring the potential for precision medicine.},
}

*Pancreatic Neoplasms/drug therapy/pathology/genetics/therapy/metabolism
*Photochemotherapy/methods
Humans
Animals
*CRISPR-Cas Systems
Mice
NF-E2-Related Factor 2/genetics/metabolism
Cell Line, Tumor
*Photosensitizing Agents/chemistry/pharmacology/administration & dosage/therapeutic use
Chlorophyllides
*DNA/chemistry
Reactive Oxygen Species/metabolism
Porphyrins/chemistry/pharmacology/administration & dosage
Hemin/chemistry
Apoptosis/drug effects
Gene Editing
Ribonucleoproteins/genetics
G-Quadruplexes

@article {pmid41162706,
year = {2026},
author = {Chong, Z and Hui, S and Qiu, X and Palakurty, S and Sariol, A and Kaszuba, T and Nguyen, MN and Li, P and Raju, S and Hall, PD and Nelson, CA and Baltazar-Perez, I and Price, DA and Rothlauf, PW and Crowe, JE and Whelan, SPJ and Leung, DW and Amarasinghe, GK and Bailey, AL and Fremont, DH and Diamond, MS},
title = {Multiple LDLR family members act as entry receptors for yellow fever virus.},
journal = {Nature},
volume = {649},
number = {8095},
pages = {173-182},
pmid = {41162706},
issn = {1476-4687},
mesh = {Animals ; *Yellow fever virus/physiology/pathogenicity/metabolism ; Humans ; *Virus Internalization ; Mice ; *Receptors, LDL/metabolism/genetics/chemistry/deficiency ; *Yellow Fever/virology/metabolism ; Female ; Low Density Lipoprotein Receptor-Related Protein-1/metabolism/genetics/deficiency/chemistry ; Male ; *Receptors, Virus/metabolism ; Viral Envelope Proteins/metabolism/chemistry ; Protein Domains ; Hepatocytes/metabolism/virology ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death[1]. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR-Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4-Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1-Fc, LRP4-Fc and VLDLR-Fc decoys protected mice from YFV challenge, and LRP1-Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.},
}

Animals
*Yellow fever virus/physiology/pathogenicity/metabolism
Humans
*Virus Internalization
Mice
*Receptors, LDL/metabolism/genetics/chemistry/deficiency
*Yellow Fever/virology/metabolism
Female
Low Density Lipoprotein Receptor-Related Protein-1/metabolism/genetics/deficiency/chemistry
Male
*Receptors, Virus/metabolism
Viral Envelope Proteins/metabolism/chemistry
Protein Domains
Hepatocytes/metabolism/virology
CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid40394336,
year = {2026},
author = {Wei, Y and Gao, P and Pan, D and Li, G and Chen, Y and Li, S and Jiang, H and Yue, Y and Wu, Z and Liu, Z and Zhou, M and Chen, Y and Xu, K and Wu, Z and Wang, X},
title = {Engineering eukaryotic transposon-encoded Fanzor2 system for genome editing in mammals.},
journal = {Nature chemical biology},
volume = {22},
number = {1},
pages = {48-57},
pmid = {40394336},
issn = {1552-4469},
mesh = {Animals ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; Mice ; Humans ; CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Mammals/genetics ; HEK293 Cells ; Muscular Dystrophy, Duchenne/genetics/therapy ; },
abstract = {Eukaryotic transposon-encoded Fanzor proteins hold great promise for genome-engineering applications as a result of their compact size and mechanistic resemblance to TnpB. However, the unmodified Fanzor systems show extremely low activity in mammalian cells. Guided by the predicted structure of a Fanzor2 complex using AlphaFold3, we engineered the NlovFz2 nuclease and its cognate ωRNA to create an evolved enNlovFz2 system, with an expanded target-adjacent motif (TAM) recognition scope (5'-NMYG) and a substantially improved genome-editing efficiency, achieving an 11.1-fold increase over the wild-type NlovFz2, comparable to two previously reported IS200 or IS605 transposon-encoded TnpBs and two CRISPR-Cas12f1 nucleases. Notably, enNlovFz2 efficiently mediated gene disruption in mouse embryos and restored dystrophin expression in a humanized Duchenne muscular dystrophy mouse model with single adeno-associated virus delivery. Our findings underscore the potential of eukaryotic RNA-guided Fanzor2 nucleases as a versatile toolbox for both biological research and therapeutic applications.},
}

Animals
*Gene Editing/methods
*DNA Transposable Elements/genetics
Mice
Humans
CRISPR-Cas Systems
Endonucleases/genetics/metabolism
Mammals/genetics
HEK293 Cells
Muscular Dystrophy, Duchenne/genetics/therapy

@article {pmid41424849,
year = {2026},
author = {Liu, M and Fu, X and Zhang, H and Pan, J and Jia, Q and Zhang, C and An, F},
title = {Endothelial KSR2 regulated by genetic variation protects against atherosclerosis through AMPKα1 stabilization.},
journal = {Theranostics},
volume = {16},
number = {5},
pages = {2598-2626},
pmid = {41424849},
issn = {1838-7640},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism/pathology ; Mice ; *AMP-Activated Protein Kinases/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide/genetics ; Mice, Knockout ; Male ; Endothelial Cells/metabolism ; Diet, High-Fat/adverse effects ; Mice, Inbred C57BL ; Apolipoproteins E/genetics ; Mice, Knockout, ApoE ; Disease Models, Animal ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Apoptosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Rationale: The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (KSR2) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms. Methods: Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of KSR2 in atherosclerosis, we utilized global KSR2 knockout mice fed a high-fat diet ad libitum, pair-fed global KSR2 and Apoe (Apolipoprotein E) double knockout mice, and mice with endothelial-specific KSR2 overexpression mediated by AAV9-ICAM2. Results: Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of KSR2 expression via differential binding of the transcriptional repressor XBP1s. KSR2 expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple KSR2 gene-edited mouse models, we demonstrated that endothelial KSR2 protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A[CRBN] E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1. The subsequently activated AMPK signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects. Conclusions: Our findings provide the first comprehensive molecular explanation of the rs12822146-KSR2-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.},
}

Animals
*Atherosclerosis/genetics/metabolism/pathology
Mice
*AMP-Activated Protein Kinases/metabolism/genetics
Humans
Polymorphism, Single Nucleotide/genetics
Mice, Knockout
Male
Endothelial Cells/metabolism
Diet, High-Fat/adverse effects
Mice, Inbred C57BL
Apolipoproteins E/genetics
Mice, Knockout, ApoE
Disease Models, Animal
*Adaptor Proteins, Signal Transducing/genetics/metabolism
Apoptosis/genetics
CRISPR-Cas Systems

@article {pmid41424172,
year = {2025},
author = {Ikram, M and Farhan, M and Derakhshani, B and Kumar, S and Khan, N and Gupta, R and Usman, B and Liu, P},
title = {Machine Learning and CRISPR-Based Validation Elucidate OsWOX13 Involvement in Rice Heat Stress Tolerance and Flowering.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70714},
doi = {10.1111/ppl.70714},
pmid = {41424172},
issn = {1399-3054},
support = {YSPTZX202206//Special Project for the Academician Team Innovation Center of Hainan Province/ ; ZDYF2022XDNY185//Key Research Program of Hainan Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Machine Learning ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Heat-Shock Response/genetics ; *Flowers/physiology/genetics ; *Thermotolerance/genetics ; CRISPR-Cas Systems/genetics ; Proteomics ; },
abstract = {Rice (Oryza sativa) is a staple food for billions of people globally, but it faces significant yield losses due to heat stress. However, rice responses to heat stress remain understudied as compared to other stress factors. In this study, we combined meta-transcriptomics, machine learning, functional validation, and proteomic analysis to identify the key genes involved in heat stress tolerance in rice. We identified 409 meta-differentially expressed genes (meta-DEGs) between heat-tolerant and susceptible genotypes, which were associated with detoxification, oxidative stress, protein folding, phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. We trained five machine learning models, of which Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) outperformed the others. Using SHAP analysis, the top 14 genes for each model were identified, including the OsWOX13 gene, which was detected simultaneously across both models, indicating a positive regulator and a strong candidate for heat stress tolerance. Functional validation of OsWOX13 via CRISPR/Cas9-mediated knockout (KO) confirmed its positive role in heat stress, with a delay in flowering and survival rate of ~20% compared to ~60% for WT under heat stress. Physiological and antioxidant enzymatic activities showed a significant (p ≤ 0.05) reduction in ABA accumulation levels, increased MDA accumulation, and decreased SOD and POD activities in KO lines compared to WT. The proteomic analysis identified upregulated heat shock proteins (HSF8, BIP1, BIP5, and HSP81-1) and downregulated flowering-associated proteins (ROC6 and 4CL4) in mutant lines in response to heat stress. These results indicate that OsWOX13 enhances heat tolerance by regulating ABA signaling and antioxidant defense mechanisms. Taken together, this study highlights the efficiency of machine learning models in the identification of stress responsive genes and provides OsWOX13 as a strong positive candidate for heat stress tolerance and breeding climate-resilient rice varieties.},
}

*Oryza/genetics/physiology
*Machine Learning
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Heat-Shock Response/genetics
*Flowers/physiology/genetics
*Thermotolerance/genetics
CRISPR-Cas Systems/genetics
Proteomics

@article {pmid41423823,
year = {2025},
author = {Stamilla, A and Recchia, D and Stelitano, G and Maci, L and Marturano, MC and De Rossi, E and Chiarelli, LR and Pasca, MR and Degiacomi, G},
title = {Uncovering Insights Into the Biology of Mycobacterium tuberculosis Using Genetic Tools.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70206},
pmid = {41423823},
issn = {2045-8827},
support = {20205B2HZE_003//University of Pavia; Italian Ministry of University and Research/ ; 2022JTPP53//University of Pavia; Italian Ministry of University and Research/ ; PE00000007//NextGenerationEUMUR PNRR/ ; INF-ACT//NextGenerationEUMUR PNRR/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/pathogenicity ; Humans ; *Genetic Engineering/methods ; Tuberculosis/microbiology ; Drug Discovery ; Genome, Bacterial ; },
abstract = {Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most challenging pathogens due to its complex physiology, diverse clinical manifestations, and growing multidrug resistance. The global rise of drug-resistant Mtb strains has prompted the search for innovative genetic and molecular strategies to accelerate drug discovery and vaccine development. Progress in Mtb research has long been hindered by its slow replication rate and impermeable cell envelope, which limit the efficacy of genetic manipulation. This review outlines methodological advances that have transformed the study of Mtb pathogenesis and drug resistance mechanisms. Traditional homologous recombination-based approaches, including allelic exchange and specialized transduction, laid the groundwork for targeted mutagenesis but were limited by low efficiency. The advent of phage-derived recombineering systems, such as the Che9c RecET, has substantially improved the precision and throughput of genetic modification. Hybrid systems such as ORBIT, which combines oligonucleotide-mediated recombineering with Bxb1 integrase, have further enabled rapid and versatile genome engineering across mycobacterial species. Parallel developments in conditional gene expression systems (e.g., the use of TetR/Pip-based promoters) have facilitated the functional analysis of essential genes and the validation of novel drug targets. The advent of CRISPR-Cas technologies has represented a paradigm shift, by enabling programmable, high-fidelity gene regulation and functional genomics even in slow-growing mycobacteria. Together, these genetic innovations are transforming Mtb research by accelerating drug discovery and vaccine design, and shedding light on host-pathogen interactions.},
}

*Mycobacterium tuberculosis/genetics/drug effects/pathogenicity
Humans
*Genetic Engineering/methods
Tuberculosis/microbiology
Drug Discovery
Genome, Bacterial

@article {pmid41422267,
year = {2025},
author = {Fair, T and Pavlovic, BJ and Swope, D and Castillo, OE and Schaefer, NK and Pollen, AA},
title = {Mapping cis- and trans-regulatory target genes of human-specific deletions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11380},
pmid = {41422267},
issn = {2041-1723},
support = {DP2MH122400-01//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; F31 HG011569-01A1//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; Pan troglodytes/genetics ; Animals ; CRISPR-Cas Systems ; *Sequence Deletion/genetics ; Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Genome, Human ; Chromatin/metabolism/genetics ; Cell Proliferation/genetics ; Brain/metabolism ; },
abstract = {Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.},
}

Humans
Pan troglodytes/genetics
Animals
CRISPR-Cas Systems
*Sequence Deletion/genetics
Gene Expression Regulation
RNA, Guide, CRISPR-Cas Systems/genetics
Pluripotent Stem Cells/metabolism
Genome, Human
Chromatin/metabolism/genetics
Cell Proliferation/genetics
Brain/metabolism

@article {pmid41420495,
year = {2025},
author = {Pan, G and Wang, L and Zhu, H and Wang, H and Zheng, Z},
title = {Utilization of Miniature CRISPR-AsCas12f1 Nuclease for Efficient Genome Editing in Bacillus subtilis.},
journal = {Biotechnology journal},
volume = {20},
number = {12},
pages = {e70168},
doi = {10.1002/biot.70168},
pmid = {41420495},
issn = {1860-7314},
support = {S2023n06020216//Anhui Province Key Research and Development Plan/ ; 2019YFA0904304//National Key Research and Development Program of China/ ; YZJJ2024QN37//Hefei Institutes of Physical Science, Chinese Academy of Sciences Director's Fund/ ; },
mesh = {*Bacillus subtilis/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial ; },
abstract = {To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.},
}

*Bacillus subtilis/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Gene Knockout Techniques
*Endonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genome, Bacterial

@article {pmid41389042,
year = {2025},
author = {Nie, YG and Zhang, HS and Su, M and Zha, CJ and Yang, K and Ying, ZM},
title = {Proximity-Inducible CRISPR/Cas12a Activity by Scaffold RNA Assembly for Sensing Applications.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28088-28097},
doi = {10.1021/acs.analchem.5c06530},
pmid = {41389042},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Breast Neoplasms/diagnosis ; *RNA/chemistry/genetics/metabolism ; Adenosine Triphosphate/analysis ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Female ; *Bacterial Proteins/metabolism ; },
abstract = {The programmability and flexibility of the RNA-directed CRISPR/Cas12a system underpin its utility as a potent tool for diagnostic applications. However, existing engineered crRNA strategies are still limited by a narrow target range, inadequate specificity, and operational complexity. To overcome these challenges, a proximity-assembly and activate (PAA) strategy was developed, employing split dumbbell activators with terminally modified target-binding modules that reassemble on scaffold RNA to reconstruct functional crRNA and activate Cas12a trans-cleavage activity. The design allows universal detection of both nucleic acid and non-nucleic acid targets. Notably, owing to its strict target dependency, the assembled crRNA biosensor significantly reduces background signal and suppresses nonspecific leakage. We demonstrated that the PAA system facilitates rapid and highly specific detection of miRNA-21, ATP, and anti-Dig antibody in complex matrices, enabling single-base discrimination among miRNA variants. Moreover, the platform successfully detected endogenous miRNA-21 in serum and cellular samples from breast cancer patients, clearly distinguishing them from healthy controls. This work presents a modular, plug-and-play, and versatile platform for molecular diagnostics, holding considerable potential for advancing clinical diagnostics and precision medicine.},
}

Humans
*MicroRNAs/analysis/genetics/blood
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Breast Neoplasms/diagnosis
*RNA/chemistry/genetics/metabolism
Adenosine Triphosphate/analysis
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism/genetics
Female
*Bacterial Proteins/metabolism

@article {pmid41383046,
year = {2025},
author = {Zeng, W and Xie, X and Yu, X and Zhang, T and Cai, T and Chen, S and Chen, W and Wang, F and Wang, L and Ma, L},
title = {Combined Use of Engineered PfAGO and LbCas12a for Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27941-27949},
doi = {10.1021/acs.analchem.5c05622},
pmid = {41383046},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Pyrococcus furiosus/enzymology ; Humans ; CRISPR-Cas Systems ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis/virology ; Limit of Detection ; *CRISPR-Associated Proteins/genetics/metabolism ; *COVID-19 Nucleic Acid Testing/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The COVID-19 pandemic has underscored the urgent need for rapid, accurate, and accessible nucleic acid detection technologies. Here, we present NAEPLS (Nucleic Acid Detection Based on Engineered PfAgo and LbCas12a with Split crRNA), a novel diagnostic platform that synergistically integrates the engineered DNA-guided nuclease PfAgo (mPfAgo) from Pyrococcus furiosus with CRISPR-Cas12a for ultrasensitive and specific pathogen detection. By leveraging mPfAgo's RNA-cleaving capability and LbCas12a's compatibility with split crRNA, NAEPLS achieved detection of SARS-CoV-2 RNA. Coupling this assay with RT-RPA enabled ultrasensitive detection, achieving a limit of detection (LOD) of 10 copies/mL. The system demonstrates single-nucleotide specificity, robustly discriminating mutations in mocked samples (such as D614G) as well as in clinical isolates (such as S371P). Notably, the compatibility with lateral flow strip visualization allows this method to be implemented as a rapid POCT platform, particularly valuable in resource-limited settings. Validation with 20 clinical samples (Ct 20-39) showed 100% concordance with RT-qPCR, including reliable detection of low viral loads (Ct > 35). This study establishes a transformative paradigm for nucleic acid diagnostics, merging the precision of PfAgo with the amplification power of CRISPR for high-performance, field-deployable pathogen detection.},
}

*SARS-CoV-2/genetics/isolation & purification
Pyrococcus furiosus/enzymology
Humans
CRISPR-Cas Systems
*RNA, Viral/analysis/genetics
*COVID-19/diagnosis/virology
Limit of Detection
*CRISPR-Associated Proteins/genetics/metabolism
*COVID-19 Nucleic Acid Testing/methods
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41381501,
year = {2025},
author = {Padilla, R and Shipman, GA and Horth, C and Gravel, M and Bareke, E and Majewski, J},
title = {H3K36 Methylation as a Guardian of Epigenome Integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11371},
pmid = {41381501},
issn = {2041-1723},
support = {PJT-183939//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)/ ; P01-CA196539//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Animals ; *Histones/metabolism/genetics ; Humans ; Mice ; Methylation ; *Epigenome/genetics ; Heterochromatin/metabolism/genetics ; Mesenchymal Stem Cells/metabolism ; Methyltransferases/metabolism/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; *Epigenesis, Genetic ; Repressor Proteins/metabolism/genetics ; Mice, Knockout ; Chromatin/metabolism ; Euchromatin/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {H3K36 methylation is a key epigenetic mark with critical roles in development and disease. Here, we systematically dissect its functions using CRISPR-engineered mouse mesenchymal stem cells lacking combinations of the five H3K36 methyltransferases, culminating in quintuple knockout cells devoid of H3K36me2/3. We show that H3K36me2 influences enhancer activity, supports the expression of their target genes, and safeguards active genes from encroachment of the repressive marks, H3K27me2/3. In addition, we find that the loss of H3K36me triggers redistribution of large heterochromatic H3K9me3 domains into euchromatin, in part mediated by SUV39H1, leading to global epigenomic remodelling, constitutive heterochromatin erosion, and a collapse of 3D genome organization. Parallel analyses in human HNSCC cells overexpressing the H3K36M oncohistone reveal conserved disruptions to the epigenome and chromatin architecture. Together, these results establish H3K36 methylation as a pivotal regulator of chromatin state and genomic structure.},
}

Animals
*Histones/metabolism/genetics
Humans
Mice
Methylation
*Epigenome/genetics
Heterochromatin/metabolism/genetics
Mesenchymal Stem Cells/metabolism
Methyltransferases/metabolism/genetics
Histone-Lysine N-Methyltransferase/genetics/metabolism
*Epigenesis, Genetic
Repressor Proteins/metabolism/genetics
Mice, Knockout
Chromatin/metabolism
Euchromatin/metabolism
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid41370671,
year = {2025},
author = {Gao, Z and Lin, K and Gong, Y and Zhao, Y and Zhang, S},
title = {Core-Shell Tripeptide-Lipid/PEI Nanocarriers Enable Efficient Plasmid-Based CRISPR/Cas9 Editing of VEGFR2.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {41},
number = {50},
pages = {33716-33730},
doi = {10.1021/acs.langmuir.5c02069},
pmid = {41370671},
issn = {1520-5827},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Polyethyleneimine/chemistry ; *Plasmids/genetics/chemistry ; *Vascular Endothelial Growth Factor Receptor-2/genetics ; Animals ; *Lipids/chemistry ; *Nanoparticles/chemistry ; MCF-7 Cells ; Mice ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile genome editing technology that holds tremendous promise for the treatment of various diseases. Although several delivery technologies such as electroporation, viral vectors, and lipid nanoparticles have already shown promise in preclinical and clinical applications for hematological and neuromuscular genetic disorders, in vivo application is still restricted by the inefficient delivery of CRISPR/Cas9 components. Herein, by employing the tripeptide lipid N,N-ditetradecyloxyamidoethyl trimeric ornithine amide (CDO) and polyethylenimine (PEI), we constructed novel ternary systems (pDNA/PEI/CL) for the delivery of pDNA encoding Cas9 and single-guide RNAs (sgRNAs) targeting the VEGFR2 gene. The pDNA/PEI/CL delivery systems were fabricated by condensing pDNA with PEI, followed by coating with cationic liposomes composed of CDO. This system demonstrated high transfection efficiency, successfully delivering CRISPR/Cas9 to A549 and MCF-7 cells with efficiencies of up to 91.0% (n = 3, P < 0.001), while also exhibiting lower cytotoxicity. Notably, the sgRNA1/P1/C1 complex achieved higher genome editing efficiencies than sgRNA3/P1/C1, with 38.6% vs 31.0% in A549 cells (n = 3, P < 0.01) and 26.45% vs 20.18% in MCF-7 cells (n = 3, P < 0.01). Western blot analysis showed that VEGFR2 expression decreased by 48.1% in A549 and 44.3% in MCF-7 cells, while PI3K levels were reduced by 39.6% and 42.8%, respectively. This suppression of the PI3K/Akt signaling pathway led to cell cycle arrest, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. Furthermore, animal experiments validated the antitumor efficacy, highlighting the translational potential of this platform in cancer therapy. Collectively, these findings highlight the potential of the ternary complex system as a robust and biocompatible CRISPR/Cas9 delivery strategy, offering a promising avenue for gene therapy in cancer and other genetic diseases.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Polyethyleneimine/chemistry
*Plasmids/genetics/chemistry
*Vascular Endothelial Growth Factor Receptor-2/genetics
Animals
*Lipids/chemistry
*Nanoparticles/chemistry
MCF-7 Cells
Mice

@article {pmid41360396,
year = {2025},
author = {Jiang, Q and Ramachandran, A and Avaro, AS and Huyke, DA and Santiago, JG},
title = {Reaction Kinetics of CRISPR trans-Cleavage Controlled Using Isotachophoresis.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27646-27653},
doi = {10.1021/acs.analchem.5c04301},
pmid = {41360396},
issn = {1520-6882},
mesh = {*Isotachophoresis/methods ; Kinetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-based diagnostics are powerful tools for nucleic acid detection due to their high specificity and programmability. However, assay sensitivity is often limited by the slow kinetics of the trans-cleavage reaction, which typically proceeds at a rate of ∼0.1 to 1 turnover per second. Here, we present a reaction-transport model and experimental study that analyze and accelerate this limiting step using electric-field-driven isotachophoresis (ITP). Building on the work of Ramachandran and Santiago, we develop a model that captures the coupling among ITP focusing, mixing, and preconcentration with CRISPR enzymatic reaction kinetics. Our analysis identifies two key regimes in ITP-coupled CRISPR reactions and derives analytical approximations for the limiting behaviors in each. Compared to a standard, well-mixed assay, we predict a 10- to 100-fold reduction in reaction duration using ITP. We validate the model with experiments across a range of target concentrations. Our work offers a quantitative framework for understanding and optimizing CRISPR trans-cleavage dynamics and provides guidance to design assays that use electric-field-mediated transport.},
}

*Isotachophoresis/methods
Kinetics
*CRISPR-Cas Systems

@article {pmid41358836,
year = {2025},
author = {Li, Q and Xu, J and Jiang, J and Gong, L and Mao, X and Wang, F and Yao, P},
title = {Nucleic acid detection method for Chlamydia psittaci based on RPA-CRISPR/Cas12a.},
journal = {Letters in applied microbiology},
volume = {78},
number = {12},
pages = {},
doi = {10.1093/lambio/ovaf138},
pmid = {41358836},
issn = {1472-765X},
support = {BE2023694//Key Research and Development Project of Jiangsu Province/ ; H2023060//Jiangsu Provincial Commission of Health Project/ ; K2024003//Key Project of the Jiangsu Provincial Commission of Health/ ; Ym2023015//Jiangsu Province Preventive Medicine Research Project/ ; Ym2023073//Jiangsu Province Preventive Medicine Research Project/ ; x202339//Jiangsu Provincial Blood Parasite and Endemic Disease Prevention Research Project of China/ ; CE20225041//Changzhou science and technology Foundation/ ; CJ20253132//Changzhou science and technology Foundation/ ; CJ20253133//Changzhou science and technology Foundation/ ; CPHM202401//Nanjing Medical University/ ; CPHM202303//Nanjing Medical University/ ; },
mesh = {*Chlamydophila psittaci/genetics/isolation & purification ; Humans ; *Psittacosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In recent years, misdiagnosis or delayed diagnosis of Chlamydia psittaci (C. psittaci) infections has led to frequent outbreaks of severe public health events, such as severe pneumonia and respiratory distress, drawing increasing attention. Rapid and simple detection methods are vital for early intervention to reduce severity and mortality. In this study, we designed highly specific RPA primers and crRNA (CRISPR RNA) based on the highly conserved CPSIT_0429 gene in the C. psittaci genome, and preliminarily established a nucleic acid detection method for C. psittaci using the RPA-CRISPR/Cas12a system. In the two-step assay, the combination of the CPSIT_0429-F1/R1 primer pair and CPSIT_0429-crRNA2 achieved a detection limit of 2 × 10° copies/μL. Incorporating 20% glycerol enabled a one-tube assay with a limit of 2 × 102 copies/μL. Furthermore, the method showed no cross-reactivity with common respiratory pathogens such as influenza virus, SARS-CoV-2, and Streptococcus pneumoniae, demonstrating excellent specificity. Both the two-step and one-tube methods were compared with qPCR-verified C. psittaci positive samples. The results indicated that both assays showed high consistency with qPCR results. The RPA-CRISPR/Cas12a detection method is rapid, accurate, highly sensitive, and specific, providing a reliable platform for early diagnosis and clinical management of C. psittaci infections.},
}

*Chlamydophila psittaci/genetics/isolation & purification
Humans
*Psittacosis/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41344241,
year = {2026},
author = {Peng, Y and Xu, J and Chen, B and Zeng, D and Yu, X and Chen, W},
title = {Ultrasensitive detection of lead ion in tea samples using a versatile and robust multi-DNAzyme DNA machine mediated CRISPR/Cas12a signal amplification system.},
journal = {Food chemistry},
volume = {499},
number = {},
pages = {147352},
doi = {10.1016/j.foodchem.2025.147352},
pmid = {41344241},
issn = {1873-7072},
mesh = {*DNA, Catalytic/genetics/chemistry ; *Tea/chemistry ; *Lead/analysis ; *Food Contamination/analysis ; CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Camellia sinensis/chemistry/genetics ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Ions/analysis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Lead ion (Pb[2+]) contamination in tea poses serious threats to food safety. We present a novel detection platform integrating a multifunctional DNAzyme machine with the CRISPR/Cas12a system. Upon target Pb[2+] binding, the multi-DNAzyme activates the DNA machine, initiating multiple isothermal cycles that generate a poly-A sequence. This sequence activates Cas12a, triggering trans-cleavage of the hairpin fluorescent probes and significant signal amplification. The self-sustained amplification of DNA machine eliminates the need for any auxiliary probes, greatly simplifying the assay design. This platform achieves excellent sensing perfromances with detection limit of 67.53 pM, wide linear range (0.01-100 nM), satisfied specificity and high recovery rates above 96 % in real tea samples. This integration of DNAzyme recognition with CRISPR/Cas12a signal enhancement provides a rapid, and cost-effective method for Pb[2+] detection. This strategy offers practical potential for food safety monitoring and environmental assessment, and future research may expand its applicability to other heavy metal contaminants.},
}

*DNA, Catalytic/genetics/chemistry
*Tea/chemistry
*Lead/analysis
*Food Contamination/analysis
CRISPR-Cas Systems
*Biosensing Techniques/methods/instrumentation
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*Camellia sinensis/chemistry/genetics
*Endodeoxyribonucleases/genetics/chemistry/metabolism
Ions/analysis
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41288355,
year = {2025},
author = {Xie, W and Cai, Z and Bao, Z},
title = {Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {12},
pages = {e0161825},
doi = {10.1128/aem.01618-25},
pmid = {41288355},
issn = {1098-5336},
support = {2023YFF1204500//National Key Research and Development Program of China/ ; 22308316//National Natural Science Foundation of China/ ; 226-2025-00043, 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Genetic Engineering/methods ; Benchmarking ; Genetic Variation ; },
abstract = {Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.IMPORTANCECRISPR/Cas9 has facilitated yeast functional genomics by generating a large number of precise genetic variants in a very short period of time. This enabled the interrogation of reconstituted natural genetic variants across different genetic backgrounds or entirely synthetic mutations to discover novel or improved functions. However, Cas9 only targets a limited genomic sequence space due to its preference for G-rich PAM sequences. In this study, we close this gap by developing a CRISPR/Cas12a-based system to engineer user-defined genetic variants targeting T-rich PAM sequences. Our system adopts a homology-integrated design and the most PAM-relaxed Cas12a characterized in yeast to date. These features collectively enabled the creation of genetic variant libraries and multiplex edited strains. This genome editing tool can be used together with Cas9-based tools to interrogate a greater genomic sequence space.},
}

*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Genetic Engineering/methods
Benchmarking
Genetic Variation

@article {pmid41195558,
year = {2025},
author = {Mohammadi, R and Sakic, I and Jain, A and Mathur, P and Weiss, T and deMello, AJ and Stavrakis, S and Asghari, M},
title = {Ultra-High-Throughput Viscoelastic Squeezing for Mechanoporation and Efficient Intercellular Delivery.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {51},
pages = {e07981},
doi = {10.1002/smll.202507981},
pmid = {41195558},
issn = {1613-6829},
mesh = {Viscosity ; Humans ; *Elasticity ; *Electroporation/methods ; Microfluidics/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {Cell-based therapies have transformed the treatment landscape for a range of diseases, leveraging both genome modification and cell reprogramming to create targeted treatments. Such therapies rely on the efficient internalization of biomolecules into living cells. Unfortunately, existing cargo delivery methods, such as those based on viral vectors and electroporation, are often compromised by cytotoxicity, poor delivery efficiencies, and low throughput. To overcome these limitations, a viscoelastic squeezing methodology is presented that uses viscoelastic microfluidics to perform mechanoporation in a rapid and contact-free manner. Through the control of the flow rates of a sample stream containing cells and cargo and a surrounding viscoelastic sheath flow, the width of a "virtual channel" formed between the two streams can be regulated. Elastic forces generated within this virtual channel are then used to deform contained cells and internalize user-defined payloads. The effectiveness and utility of the platform are assessed through the delivery of mRNA, plasmid DNA, and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) ribonucleoprotein complexes into a variety of cell lines. Data confirms that viscoelastic squeezing provides for enhanced delivery efficiencies when compared to conventional poration techniques, whilst maintaining high cell viabilities and throughputs of 20 million cells per minute, and thus represents a powerful tool for cellular engineering.},
}

Viscosity
Humans
*Elasticity
*Electroporation/methods
Microfluidics/methods
CRISPR-Cas Systems/genetics

@article {pmid41071356,
year = {2025},
author = {Asaoka, Y and Tarumoto, S and Hirose, Y and Iwamoto, A and Tokunaga, M and Matsuura, T and Takemoto, Y and Yamashita, H and Furutani-Seiki, M and Sugahara, K},
title = {LOXHD1b knockout alters swimming behavior in zebrafish.},
journal = {Cell and tissue research},
volume = {402},
number = {3},
pages = {243-254},
pmid = {41071356},
issn = {1432-0878},
support = {23H04710//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; 20K09733//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; },
mesh = {Animals ; *Zebrafish/physiology/genetics ; *Swimming/physiology ; *Zebrafish Proteins/metabolism/genetics ; *Gene Knockout Techniques ; Lateral Line System/metabolism ; Hair Cells, Auditory/metabolism ; CRISPR-Cas Systems/genetics ; Humans ; Larva ; },
abstract = {Since human inner ear hair cells do not regenerate, the current treatments of hereditary deafness depend on hearing aids or cochlear implant. However, uncovering the functions of genes responsible for hereditary hearing loss is not only useful for their diagnosis but also for developing therapies. The pathogenetic mechanism of human non-syndromic deafness DFNB77 without morphological defects in the inner year caused by LOXHD1 mutations is not fully understood. We introduced zebrafish because the lateral line hair cells are structurally and physiologically similar to the human inner ear hair cells and mutations involved in non-symptomatic hearing loss can be assessed by their swimming behavior. The knock-out (KO) of LOXHD1b gene which is expressed in the lateral line hair cells was generated using the CRISPR-Cas9 system in zebrafish, and its morphological and functional changes were evaluated. As with human patients the LOXHD1b KO zebrafish larvae did not exhibit detectable morphological defects, but showed prolonged water flow sensing time. These results suggest that LOXHD1b plays pivotal roles for the hair cell neural activity and its KO zebrafish mutant serves as a useful model for revealing the molecular mechanisms linking LOXHD with hair cell function and for a drug screening to rescue the swimming phenotype.},
}

Animals
*Zebrafish/physiology/genetics
*Swimming/physiology
*Zebrafish Proteins/metabolism/genetics
*Gene Knockout Techniques
Lateral Line System/metabolism
Hair Cells, Auditory/metabolism
CRISPR-Cas Systems/genetics
Humans
Larva

@article {pmid40402429,
year = {2025},
author = {Shang, Z and Liu, S and Liu, D and Wang, Y and Pei, X and Li, S and He, Y and Tong, Y},
title = {Systematically Investigating CRISPR/Cas12a Fluorescent Biosensor for Sensitive and Specific Single Nucleotide Variants Detection.},
journal = {Journal of fluorescence},
volume = {35},
number = {11},
pages = {11081-11089},
pmid = {40402429},
issn = {1573-4994},
support = {(No. 22004005).//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques/methods ; *SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; Humans ; COVID-19/diagnosis/virology ; Fluorescent Dyes/chemistry ; Fluorescence ; Mutation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Precise identification and detection of single nucleotide variation (SNV) concomitant with excess wild-type DNA is greatly needed for invasive disease diagnosis, pathogens detection and early prediction of drug responsiveness. Many variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), notably the D614G and N501Y mutations, have been shown to significantly increase the infectivity of pandemics. We herein investigated CRISPR/Cas12a integrated three types fluorescent reporters and two crRNAs for SNV detection by taking D614G and N501Y variants of SARS-CoV-2 as model examples. We systematically screened all possible base substitutions from positions 0 to 19 and identified the middle position of crRNA could efficiently increase the specificity from both theoretical and experimental standpoints. With selected mutation location of crRNA, we then investigated the specificity of ssDNA, dsDNA and molecular beacon (MB) fluorescent reporters and proved the MB reporters can efficiently increase the discriminatory factors. Furthermore, we designed an additional mutation site on crRNA to increase the specificity. For user convenience, we engineered the lateral flow strips to present the results visualized with the naked eyes. Results of specific variants from Omicron proved the feasibility of clinical applications. These findings indicated that the proposed method is a powerful tool for monitoring the key mutations in pathogens and allows for modifications to incorporate newer upcoming variants.},
}

*Biosensing Techniques/methods
*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide
Humans
COVID-19/diagnosis/virology
Fluorescent Dyes/chemistry
Fluorescence
Mutation
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41419797,
year = {2025},
author = {Das, IS and Shi, Q and Dreischhoff, S and Polle, A},
title = {Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-025-07955-z},
pmid = {41419797},
issn = {1471-2229},
abstract = {BACKGROUND: Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels.

RESULTS: Using in-silico database searches, we identified 28 KTIs in the haploid genome of Populus x canescens; 21 of them were responsive to herbivory. The greatest induction by herbivory was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri.017G153200 orthologue). Mechanical wounding and methyl-jasmonate treatments resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE, a key enzyme involved in jasmonate biosynthesis. On the contrary, exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR-Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400 + kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced, and their double knockout lines in increased weight gain of the larvae. In contrast, overexpressing or knockout lines of KTI_53200 had no effect on larval weight gain compared with controls.

CONCLUSION: KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against H. armigera in trees and presents new options to investigate growth-defense theories.},
}

@article {pmid41418951,
year = {2025},
author = {Chen, J and Bian, X and Zheng, X and Peng, B and Li, R and Du, H and Zhou, L and Wen, Y},
title = {The synergistic effect of DNA nanostructures and CRISPR/Cas system for cancer diagnosis and treatment.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {149741},
doi = {10.1016/j.ijbiomac.2025.149741},
pmid = {41418951},
issn = {1879-0003},
abstract = {Cancer remains one of the most formidable global public health challenges, exerting a profound and detrimental impact on human health. Despite substantial advancements in cancer research, the escalating incidence and mortality rates underscore the persistent and growing burden on global healthcare systems. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, heralded as a revolutionary gene-editing tool, holds immense promise for cancer treatment. However, its efficacy is critically contingent upon developing efficient delivery strategies. DNA nanocarriers, characterized by their programmability, sequence specificity, and design flexibility, emerge as a highly effective vehicle for delivering the CRISPR/Cas system, facilitating the precise transportation of gene-editing tools to the cell nucleus. The integration of DNA nanocarriers with CRISPR/Cas technology provides a new paradigm for precise and controllable gene editing. Through programmable spatial assembly, DNA nanocarriers can protect Cas9 ribonucleoprotein complexs (RNPs), facilitate endosomal escape, and co-localize donor DNA to promote homology-directed repair. These synergistic effects bridge molecular programmability and genetic functionality, paving the way for safer and more efficient genome engineering. This review aims to evaluate the application of DNA nanocarriers in cancer diagnosis comprehensively and to explore their potential utility in cancer therapy when combined with the CRISPR/Cas system, offering novel insights and significant scientific contributions to the field.},
}

@article {pmid41418774,
year = {2025},
author = {Ji, R and Chen, Q and Zhang, Y},
title = {Emerging trends in gene and cell therapy: CRISPR in DNA editing and beyond.},
journal = {Cell reports. Medicine},
volume = {},
number = {},
pages = {102459},
doi = {10.1016/j.xcrm.2025.102459},
pmid = {41418774},
issn = {2666-3791},
abstract = {CRISPR-based gene and cell therapies are rapidly transitioning from experimental platforms to clinical reality, exemplified by the recent approval of CRISPR-derived treatments for β-hemoglobinopathies. This review highlights how advances in genome editing technologies, ranging from CRISPR-Cas nucleases to base and prime editors, are expanding the therapeutic landscape beyond traditional gene knockout approaches. We focus on the clinical translation of these tools, drawing on examples from ongoing and completed human trials to illustrate their potential across diverse disease areas. Furthermore, we discuss critical considerations such as delivery challenges, long-term safety, immune responses, and editing specificity, all of which are critical to the safe and effective integration of CRISPR technologies into modern medicine.},
}

@article {pmid41417901,
year = {2025},
author = {Chammas, P and Xie, SQ and Sepulveda-Rincon, LP and Leeke, BJ and Dore, MH and Dormann, D and Wagner, RT and Chang, N and Jones, PL and McManus, MT and Karimi, MM and Young, G and Percharde, M},
title = {CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency, and cell death.},
journal = {Science advances},
volume = {11},
number = {51},
pages = {eadu9092},
pmid = {41417901},
issn = {2375-2548},
mesh = {Animals ; Mice ; Cell Death/genetics ; *Homeodomain Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *DNA Transposable Elements/genetics ; Zygote/metabolism ; },
abstract = {Transposable elements (TEs) are powerful cis-regulatory drivers of gene expression, particularly during early development when many TEs become de-repressed. MERVL elements are transiently up-regulated in mouse totipotent two-cell (2C) embryos during major zygotic genome activation (ZGA) and 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos. Moreover, sustained Dux activation causes cell toxicity, which may or may not be linked to MERVL. Using a CRISPR activation system, we unpick the relative role of Dux and MERVL in ZGA, totipotent-like characteristics, and cell toxicity. We find that MERVL activation drives a portion of the Dux-dependent transcriptome, sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves the proapoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a previously unknown player in Dux-driven pathology.},
}

Animals
Mice
Cell Death/genetics
*Homeodomain Proteins/genetics/metabolism
*CRISPR-Cas Systems
Gene Expression Regulation, Developmental
*Transcription Factors/genetics/metabolism
*DNA Transposable Elements/genetics
Zygote/metabolism

@article {pmid41417296,
year = {2025},
author = {Rehman, T and Sharif, A and Khalid, L and Sajid, I},
title = {Whole genome sequencing and genomic characterization of the extensively drug-resistant Acinetobacter baumannii recovered from clinical samples in Lahore, Pakistan.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {217},
pmid = {41417296},
issn = {1573-4978},
mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation & purification/pathogenicity ; Pakistan ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing/methods ; *Acinetobacter Infections/microbiology/genetics/drug therapy ; Genome, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Extensively drug-resistant (XDR) Acinetobacter baumannii has turned into a significant nosocomial pathogen often resistant to all available classes of antibiotics. It has been identified as a key public health issue due to its capacity to get resistance determinants, virulence genes, and mobile genetic elements. This study hypothesized that the XDR A. baumannii isolated in Pakistan would harbor some of the key genomic determinants of the resistance and pathogenicity.

METHODOLOGY: A total of 11 A. baumannii were obtained from the patient samples and were identified using microbiological, biochemical, and genomic analysis. The sensitivity to antibiotics was determined by Kirby-Bauer disc diffusion assay. While the whole genome sequencing (WGS) was performed for the single XDR Acinetobacter isolate TAB-4 and its in-silico genome analysis on resistant genes, virulence factors, plasmids, mobile genetic elements, prophages, CRISPR-Cas and biosynthetic gene clusters was conducted.

RESULTS: The isolated strains were 10 multidrug resistant (MDR) and one (TAB-4) was XDR Acinetobacter strain. The AST showed resistance to nearly all classes of antibiotics, and with limited susceptibility to tetracyclines and aminoglycosides. The genome of TAB-4 strain comprised of 3.94 Mb, 120 contigs, and a GC content of 39.14%. The major resistant determinants found were blaOXA-23, blaOXA-69, blaNDM-1, blaADC-25, aminoglycoside modifying enzymes, and efflux pumps (adeABC, adeFGH, adeIJK). Virulence-associated genes (ompA, bap, csuA/B-E, plc) were identified along with five prophage regions, multiple CRISPR arrays, and a betalactone biosynthetic gene cluster.

CONCLUSION: This study to the best of our knowledge reports the first detailed WGS-based characterization of an XDR A. baumannii from Lahore, Pakistan. These genomic findings offer significant insights into the resistance and virulence factors underlying this challenging clinical issue. Hence, there is an urgent need to find new or alternative treatment methods against high-risk pathogens like XDR A. baumannii.},
}

*Acinetobacter baumannii/genetics/drug effects/isolation & purification/pathogenicity
Pakistan
Humans
*Drug Resistance, Multiple, Bacterial/genetics
Whole Genome Sequencing/methods
*Acinetobacter Infections/microbiology/genetics/drug therapy
Genome, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Virulence Factors/genetics
Genomics/methods
Microbial Sensitivity Tests

@article {pmid41416522,
year = {2025},
author = {Stella, G and Ye, L and Brady, SF and Marraffini, L},
title = {CARF-HAD phosphatase effectors provide immunity during the type III-A CRISPR-Cas response.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41416522},
issn = {1362-4962},
support = {R01GM149834/GF/NIH HHS/United States ; R35GM122559/GF/NIH HHS/United States ; //HHMI/ ; },
mesh = {*CRISPR-Cas Systems ; *Phosphoric Monoester Hydrolases/metabolism/genetics/chemistry ; Protein Domains ; Adenosine Triphosphate/metabolism ; Escherichia coli/genetics/virology ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here, we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain. We named these proteins Chp for CRISPR-associated HAD phosphatase. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP, and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP, and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.},
}

*CRISPR-Cas Systems
*Phosphoric Monoester Hydrolases/metabolism/genetics/chemistry
Protein Domains
Adenosine Triphosphate/metabolism
Escherichia coli/genetics/virology
Adenine Nucleotides/metabolism
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Bacterial Proteins/metabolism/chemistry/genetics

@article {pmid41415454,
year = {2025},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2025.12.05.692657},
pmid = {41415454},
issn = {2692-8205},
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia . However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A , DNMT3B , PERIOD2 , and DMRT1 in Daphnia magna . Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}

@article {pmid41415442,
year = {2025},
author = {Sabol, AL and Mengiste, AA and Sreekanth, V and Singh, P and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, K and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2025.12.11.693673},
pmid = {41415442},
issn = {2692-8205},
abstract = {Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}

@article {pmid41414673,
year = {2025},
author = {Chan, J and Wu, Z and Liu, M and Wang, T and Liu, H and Cao, R and Li, X and Li, X and Zhan, S and Cheng, J and Xu, Y and He, M and Feng, Y and Xu, Q and Sun, Y and Chen, L and Hu, P},
title = {Systematic enhancer mapping and functional analysis in zebrafish with optimized CRISPR interference.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414673},
issn = {1362-4962},
support = {32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; (32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Enhancer Elements, Genetic ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Noncoding cis-regulatory elements, particularly enhancers, are crucial for controlling gene expression. However, the in vivo use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) to study enhancer function has been limited in zebrafish, which is widely used in early development and human disease research. Here, we optimized the CRISPRi system in zebrafish to achieve efficient suppression of tyr expression by fine-tuning component concentrations. Applying this optimized system, we functionally annotated distal enhancers of globin genes. Using Hi-C and histone modification assays, we systematically mapped 434 enhancer-promoter (EP) interactions across the genome. Among these EP loops, CRISPRi perturbation identified previously unreported enhancers with regulatory strengths surpassing known elements, demonstrated by disrupted phenotypes in fin and blood cell development. Additionally, several unreported EP loops were validated, underscoring the robustness of our integrated approach. This study not only provides an optimized CRISPRi system for zebrafish but also introduces a powerful platform that integrates computational and experimental strategies for advancing cis-regulatory element annotation in vertebrate gene regulation.},
}

Animals
*Zebrafish/genetics
*Enhancer Elements, Genetic
*CRISPR-Cas Systems
Promoter Regions, Genetic
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41414668,
year = {2025},
author = {Semsey, S and Søndberg, E and Røen, M and Hallström, B and Petersen, AØ and Alfastsen, L and Bosch, BR and Wohl, B and Clube, J and van der Helm, E and Groendahl, C and Mougiakos, I},
title = {Characterization and engineering of a type IV-A3 CRISPR-Cas system for genome editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414668},
issn = {1362-4962},
support = {//SNIPR Biome/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Klebsiella pneumoniae/genetics ; Genome, Bacterial ; DNA Helicases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have revolutionized genome engineering technologies, but type IV CRISPR-Cas systems and their genome engineering potential have been critically underexplored. In this study, we identified a type IV-A3 CRISPR-Cas system from a clinical Klebsiella pneumoniae isolate and characterized its plasmid targeting activity and capacity to suppress chromosomal and plasmid gene expression in Escherichia coli. We revealed the pivotal role of Csf3 (Cas5) and the dispensable roles of Csf1 (Cas8-like) and Csf4 (DinG helicase) subunits in IV-A3 CRISPR-Cas complex formation. The system prevents plasmid propagation via interplay between DinG helicase activity and strategic protospacer positioning relative to plasmid replication and maintenance components. We enabled the IV-A3 CRISPR-Cas system to introduce lethal, sequence-specific double-stranded (ds)DNA breaks in the E. coli chromosome by fusing the nuclease domain of the I-TevI nuclease to the Cas8 N-terminus. Further, we developed a series of base editors, with various editing efficiencies and windows, by fusing the PmCDA1 cytidine deaminase to the Cas8, Cas5, and DinG subunits. Finally, conjugative transfer of the Cas5-PmCDA1 base editor into E. coli deactivated the tryptophan repressor gene, boosting IAA production. Our study provides new insights into type IV-A3 CRISPR-Cas systems and highlights their potential in genome engineering applications.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
*Gene Editing/methods
Plasmids/genetics
Klebsiella pneumoniae/genetics
Genome, Bacterial
DNA Helicases/genetics/metabolism
Bacterial Proteins/genetics/metabolism

@article {pmid41414667,
year = {2025},
author = {Klein, N and Sanchez-Londono, M and Kara, MM and Gomes-Filho, JV and Novak, S and Kholeif, KH and Pekarek, L and Caliskan, N and Randau, L},
title = {Type I-Fv and engineered type IV-A1 CRISPR-Cas effectors facilitate genome reduction in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414667},
issn = {1362-4962},
support = {//German Research Foundation/ ; 360987069//DFG/ ; 505997786//DFG/ ; //Microcosm Earth Center/ ; INST 93/1021-1 FUGG//Helmholtz Association/ ; //Philipps-Universität Marburg/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; DNA Repair ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Class 1 CRISPR-Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR-Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR-Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR-Cas-mediated genome editing.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
*Gene Editing/methods
*Genome, Bacterial
DNA Repair
CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins/genetics/metabolism

@article {pmid41274609,
year = {2026},
author = {Lau, CH and Li, X and Liang, QL and Guo, R and Ren, X and Xu, Z and Zhu, Y and Lai, Y and Liu, G and Huang, Y and Wu, W and Zhu, H and Chen, J and Zhang, X},
title = {CRISPR technology for diagnosis and treatment of human brucellosis.},
journal = {Journal of microbiological methods},
volume = {240},
number = {},
pages = {107339},
doi = {10.1016/j.mimet.2025.107339},
pmid = {41274609},
issn = {1872-8359},
mesh = {Humans ; *Brucellosis/diagnosis/therapy/drug therapy/microbiology ; Animals ; *Brucella/genetics/isolation & purification/drug effects ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Livestock/microbiology ; Point-of-Care Testing ; Anti-Bacterial Agents/therapeutic use ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The global burden of Brucella infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of Brucella in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the Brucella replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant Brucella strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global Brucella infections.},
}

Humans
*Brucellosis/diagnosis/therapy/drug therapy/microbiology
Animals
*Brucella/genetics/isolation & purification/drug effects
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Livestock/microbiology
Point-of-Care Testing
Anti-Bacterial Agents/therapeutic use
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41413028,
year = {2025},
author = {Chen, Q and Jiang, X and Yang, B and Deng, Z and Sun, Y},
title = {Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-025-66237-2},
pmid = {41413028},
issn = {2041-1723},
abstract = {Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.},
}

@article {pmid41412367,
year = {2025},
author = {Jose, J and Hamow, KÁ and Éva, C and Moncsek, B and Kyrpa, T and Reinoso, LG and Bozsó, Z and Bakonyi, J and Balázs, E and Sági, L},
title = {CRISPR/Cas-mediated polyphenol oxidase gene knockout in potato reveals divergent roles in resistance to bacterial wilt and late blight.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112944},
doi = {10.1016/j.plantsci.2025.112944},
pmid = {41412367},
issn = {1873-2259},
abstract = {Polyphenol oxidases (PPOs) play a pivotal role in plant immune responses by catalysing the oxidation of phenolic compounds into cytotoxic quinones and melanin and contributing to the fortification of cell walls. Despite their biological significance, the high expression of PPOs in potatoes is not desirable due to their promotion of tuber browning. This study elucidates the relationship between PPO activity and defense mechanisms against the diverse pathogens Ralstonia solanacearum (Rs) and Phytophthora infestans (Pi) while mitigating enzymatic browning. CRISPR/Cas-mediated editing of the tuber- and root-specific PPO genes in the 'Désirée' and 'Balatoni Rózsa' potato cultivars considerably reduced enzymatic activity and browning. Among four PPO-edited mutant lines, three exhibited increased susceptibility to Rs while responses to Pi remained unchanged, underscoring the importance of PPOs in resistance to Rs. The PPO knockouts resulted in significant shifts in metabolite and hormone profiles characterized by elevated levels of dihydrokaempferol, coniferyl alcohol and taxifolin among other metabolites in the roots of Rs-susceptible mutants. Additionally, reduced PPO activity in these lines correlated with increased concentrations of salicylic acid, jasmonic acid and several antimicrobial compounds and alterations in flavonoid regulation. These findings highlight the complex role of PPOs in plant defense, establishing a positive correlation between PPO activity and resistance to Rs, while offering insights into the trade-offs associated with PPO gene editing in potatoes.},
}

@article {pmid41412287,
year = {2025},
author = {Su, Z and Liang, Z and Wu, Q and Xu, S and Li, C and Zheng, H and Wu, C and Ji, W and Niu, Y and Yang, Z},
title = {Metal-organic frameworks for CRISPR/Cas9 gene editing delivery: Innovations in therapeutic and diagnostic applications.},
journal = {Acta biomaterialia},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.actbio.2025.12.030},
pmid = {41412287},
issn = {1878-7568},
abstract = {CRISPR/Cas gene editing technology demonstrates significant promise in the treatment of various diseases, and a precise, efficient and safe delivery system is a key to realize gene therapy. Although traditional viral vectors can achieve superior transfection efficiency, viruses suffer from low reproduction efficiency and the risk of random gene integration, further limiting their wide application. Notably, metal-organic frameworks (MOFs), with tunable pore structure, easy surface chemical modification, good biocompatibility and physiological stability, have drawn much attention in the domain of targeted delivery of gene editing systems. Compared to lipid nanoparticles (LNPs) and extracellular vesicles (EVs), MOFs offer superior cargo loading (>80 % for proteins) and protect nucleic acids from degradation, while their stimuli-responsive degradation enables controlled release. This review focus on the cutting-edge advances of intelligent-responsive MOFs in delivering gene editing systems to against diseases, including endogenous responses (e.g., ATP, pH, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Additionally, we further summarize the research of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination. This study will help facilitate the clinical translation of MOFs-based CRISPR/Cas delivery systems in the field of therapy and detection of diseases. STATEMENT OF SIGNIFICANCE: This article reviews the cutting-edge advances of intelligent-responsive MOFs in delivering CRISPR/Cas systems to against diseases, including endogenous responses (e.g., pH, ATP, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Importantly, the potential applications of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination also have been discussed. This study will provide insights for the development of MOFs-based CRISPR/Cas delivery systems in the therapy and detection of clinical diseases.},
}

@article {pmid41412110,
year = {2025},
author = {Snell, JC and Nelson, BJ and Matreyek, KA},
title = {DIALing in elevated expression setpoints with promoter shortening.},
journal = {Cell systems},
volume = {16},
number = {12},
pages = {101482},
doi = {10.1016/j.cels.2025.101482},
pmid = {41412110},
issn = {2405-4720},
mesh = {*Promoter Regions, Genetic/genetics ; *Gene Expression Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {DIAL is a novel framework for temporal control of transcript abundances in engineered cells. Targeted excision of DNA spacers in transgenic promoters permits controlled transitions of protein expression between setpoints. DIAL expands the repertoire of bioengineering tools for controlling protein expression, cell fates, and biological systems in general.},
}

*Promoter Regions, Genetic/genetics
*Gene Expression Regulation/genetics
Humans
CRISPR-Cas Systems/genetics

@article {pmid41410934,
year = {2025},
author = {Deora, S and Deora, GS and Nigam, S and Harish, },
title = {Hacking heterocysts: advances in the genetic regulation of heterocyst differentiation.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {80},
pmid = {41410934},
issn = {1432-072X},
mesh = {Nitrogen Fixation/genetics ; *Gene Expression Regulation, Bacterial ; *Cyanobacteria/genetics/metabolism/growth & development ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Heterocyst differentiation in certain filamentous cyanobacteria is a multifaceted process essential for nitrogen fixation, orchestrated by a sophisticated regulatory network that encompasses several key stages. These include induction, pattern differentiation, commitment, extracellular layer formation, cell-cell communication, and ultimately, nitrogen fixation and metabolism. Key regulators like NtcA and HetR control heterocyst development, while proteins such as PatS, HetN, and PatA modulate pattern formation. Certain non-coding RNAs, such as NsiR1, Yfr1, and NsiR4, also regulate gene expression and contribute to the shutdown of CO2 fixation in differentiating heterocysts. Meanwhile, the heterocysts' unique envelope protects nitrogenase from oxygen, enabling nitrogen fixation. Genetic engineering approaches, including CRISPR-Cas systems, have been employed to increase heterocyst frequency and enhance the production of compounds such as ethanol, butanol and H2. By manipulating genes responsible for heterocyst differentiation, scientists can optimize nitrogen fixation, develop efficient biofertilizers, and unlock opportunities for a more sustainable future in agriculture and biotechnology. This review addresses the current understanding of the regulatory networks and molecular mechanisms that influence the development and function of heterocysts, providing insights into the biology and potential applications of these specialized cells through gene manipulations.},
}

Nitrogen Fixation/genetics
*Gene Expression Regulation, Bacterial
*Cyanobacteria/genetics/metabolism/growth & development
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41410807,
year = {2025},
author = {Sarroukh, I and Ibriz, M and Yakkou, L and Lebkiri, N and Fokar, M and Iraqi, D and Gaboun, F and Diria, G and Abdelwahd, R},
title = {The Agrobacterium-mediated genetic transformation: a gateway for efficient CRISPR/Cas9 gene editing in leguminous.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {57},
pmid = {41410807},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Transformation, Genetic/genetics ; *Plants, Genetically Modified/genetics/growth & development ; *Agrobacterium tumefaciens/genetics ; },
abstract = {Climate change enhances the damaging consequences of abiotic and biotic stressors, leading to severe soil fertility loss and ecosystem degradation worldwide. Leguminous have contributed significantly to replenishing soil nitrogen via symbiotic nitrogen fixation, contributing approximately 15% of nitrogen input, which is crucial for soil health and enhancing crop production. There is an increasing integration of new biotechnological interventions, such as genome editing, including the CRISPR/Cas9 system, and transgenesis, in addition to classical breeding, to make agriculture more resilient. In this review, we examine several elements that influence the genetic transformation system employing Agrobacterium tumefaciens strains in leguminous to make it an ideal vehicle for CRISPR/Cas9 component delivery. The variables investigated in our study included the incubation period, co-cultivation duration, bacterial density, selectable marker, concentration, and growth regulators used. In addition, the selection and efficiency of the explant choice for transformation should be considered in future studies. However, there have been parallel recommendations for the gradual application of selectable markers such as kanamycin.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Transformation, Genetic/genetics
*Plants, Genetically Modified/genetics/growth & development
*Agrobacterium tumefaciens/genetics

@article {pmid41410797,
year = {2025},
author = {Appolonia, CN and Centore, JT and Shukla, S and Hluck, J and Conlon, RA and Ramakrishnan, P},
title = {A CRISPR/Cas9 assisted strategy for the conditional expression of human NF-kappaB c-Rel cDNA in mouse T cells: design, prospects, and challenges.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {56},
pmid = {41410797},
issn = {1573-9368},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Transgenic ; *Proto-Oncogene Proteins c-rel/genetics ; Promoter Regions, Genetic ; Mice, Inbred NOD ; Gene Editing ; *T-Lymphocytes/metabolism ; DNA, Complementary/genetics ; *Diabetes Mellitus, Type 1/genetics ; NF-kappa B/genetics ; },
abstract = {Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.},
}

Animals
Humans
Mice
*CRISPR-Cas Systems/genetics
Mice, Transgenic
*Proto-Oncogene Proteins c-rel/genetics
Promoter Regions, Genetic
Mice, Inbred NOD
Gene Editing
*T-Lymphocytes/metabolism
DNA, Complementary/genetics
*Diabetes Mellitus, Type 1/genetics
NF-kappa B/genetics

@article {pmid41410478,
year = {2026},
author = {Wang, X and Yu, G and Luo, Y and Chen, T and Zhang, X and Ye, L and Yang, C and Chen, Q},
title = {The autophagy-related protein PlAtg26b regulates vegetative growth, reproductive processes, autophagy, and pathogenicity in Peronophythora litchii.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2606498},
doi = {10.1080/21505594.2025.2606498},
pmid = {41410478},
issn = {2150-5608},
mesh = {*Autophagy ; Virulence ; Plant Diseases/microbiology/parasitology ; *Autophagy-Related Proteins/genetics/metabolism ; Litchi/microbiology/parasitology ; *Oomycetes/pathogenicity/growth & development/genetics ; CRISPR-Cas Systems ; Reproduction ; Plant Leaves/microbiology ; Mitochondria/metabolism ; Gene Knockout Techniques ; Hyphae/growth & development ; },
abstract = {Peronophythora litchii is an oomycete pathogen responsible for litchi downy blight, a significant threat to global litchi production. Autophagy, a conserved degradation pathway crucial for the growth, development, and pathogenicity of phytopathogenic organisms, remains an area of active investigation. In this study, we characterized the function of the Atg26 homolog PlAtg26b in P. litchii. Using the CRISPR/Cas9 genome editing system, we generated PlATG26b knockout mutants and determined that PlAtg26b localizes to mitochondria under stress conditions. Although deletion of PlATG26b did not impair selective autophagy, it markedly reduced Atg8-PE synthesis, vegetative hyphal growth, asexual and sexual reproduction, and zoospore release. Furthermore, PlATG26b-deficient mutants exhibited significantly reduced virulence on litchi fruits and leaves. Collectively, our findings demonstrate that PlAtg26b plays a pivotal role in the biological development and pathogenicity of P. litchii.},
}

*Autophagy
Virulence
Plant Diseases/microbiology/parasitology
*Autophagy-Related Proteins/genetics/metabolism
Litchi/microbiology/parasitology
*Oomycetes/pathogenicity/growth & development/genetics
CRISPR-Cas Systems
Reproduction
Plant Leaves/microbiology
Mitochondria/metabolism
Gene Knockout Techniques
Hyphae/growth & development

@article {pmid41409480,
year = {2025},
author = {Anwar, M and Vinothkanna, A and Jia, AQ},
title = {Fostering plant protection against certain bacterial diseases through quorum-sensing signal molecules: a critical review.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1602573},
pmid = {41409480},
issn = {1664-462X},
abstract = {Quorum sensing (QS) and clustered regularly interspaced short palindromic repeats (CRISPR) systems are envisaged as revolutionary in abating plant bacterial pathogens. Bacterial cell-cell communication and plant pathogen QSSMs (quorum sensing signaling molecules) are dissected for underlying mechanisms in prominent pathogens, viz., Pseudomonas syringae, Erwinia amylovora, and Xanthomonas campestris. Biofilm formation and virulence mechanisms are critically addressed to repurpose potential QS inhibition strategies. CRISPR technologies are combined with CRISPR engineering to produce enhanced disease-resistant varieties, with potential applications. QS-CRISPR interplay for deciphering the key interactive changes in plant health management is prioritized for deliberate future research outcomes. Sustainable agricultural practices are envisaged for successful lab-to-field authentic field trials and large-scale applicability across the globe. Potential technical limitations, the need for stringent agricultural laws, and future innovations are addressed. Moreover, the cost-effectiveness, enhanced crop production, yield, and productivity hindering the above key plant bacterial pathogens are comprehensively addressed against these plant bacterial pathogens. Furthermore, a future outlook characterized by extensive outreach and global implications is substantiated regardless of regional specificity, climate change, and global warming. A decade of research on advancements in adequate plant protection is revisited to incorporate augmented approaches, including artificial intelligence (AI) and machine learning, in sustainable agriculture. The significance of the present review is based on addressing QSSMs and plant protection strategies encompassing modern molecular biological techniques.},
}

@article {pmid41407671,
year = {2025},
author = {Bitew, MA and Paredes-Santos, TC and Maru, P and Krishnamurthy, S and Wang, Y and Sangaré, LO and Duley, S and Yamaryo-Botté, Y and Botté, CY and Saeij, JPJ},
title = {A genome-wide CRISPR screen identifies GRA38 as a key regulator of lipid homeostasis during Toxoplasma gondii adaptation to lipid-rich conditions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11177},
pmid = {41407671},
issn = {2041-1723},
support = {R01AI173803//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Toxoplasma/genetics/metabolism/pathogenicity/physiology ; Animals ; *Protozoan Proteins/metabolism/genetics ; Homeostasis ; Mice ; *Lipid Metabolism/genetics ; Virulence/genetics ; Phosphatidate Phosphatase/metabolism/genetics ; Toxoplasmosis/parasitology ; Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Female ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Genome, Protozoan ; },
abstract = {Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we perform a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identify the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 leads to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlate with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.},
}

*Toxoplasma/genetics/metabolism/pathogenicity/physiology
Animals
*Protozoan Proteins/metabolism/genetics
Homeostasis
Mice
*Lipid Metabolism/genetics
Virulence/genetics
Phosphatidate Phosphatase/metabolism/genetics
Toxoplasmosis/parasitology
Adaptation, Physiological/genetics
CRISPR-Cas Systems
Female
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Genome, Protozoan

@article {pmid41407557,
year = {2025},
author = {Tyagi, E and Sachan, A and Bhuyan, R and Kumari, P and Prakash, A},
title = {Next-Gen Biofilm Control: Gene Editing and Computational Approaches.},
journal = {APMIS : acta pathologica, microbiologica, et immunologica Scandinavica},
volume = {133},
number = {12},
pages = {e70122},
doi = {10.1111/apm.70122},
pmid = {41407557},
issn = {1600-0463},
mesh = {*Biofilms/drug effects/growth & development ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Phage Therapy ; Bacteriophages ; },
abstract = {Biofilms are microbial communities enclosed in an extracellular polymeric substance (EPS), significantly contributing to antimicrobial resistance (AMR) in medical, industrial, and environmental settings. Their matrix enhances microbial survival, inhibits antibiotic penetration, and facilitates horizontal gene transfer, worsening the AMR crisis. Conventional antimicrobial treatments often fail against biofilms, necessitating novel therapeutic strategies. Emerging biofilm-targeted interventions, such as nanotechnology-based antimicrobials, bacteriophage therapy, and CRISPR-Cas9 gene editing, offer promising solutions. Nanoparticles improve drug delivery, bacteriophages selectively lyse resistant bacterial populations, and CRISPR-Cas9 disrupts AMR-related genes and biofilm virulence factors. Additionally, AI and ML are advancing biofilm prediction models and antimicrobial optimization, paving the way for precision-targeted interventions. This review explores biofilm biology and next-generation biofilm control strategies, with a focus on AI-driven bioinformatics. Future research should focus on clinical translation, regulatory standardization, and scalable implementation in healthcare and industrial settings to combat biofilm-associated AMR.},
}

*Biofilms/drug effects/growth & development
*Gene Editing/methods
Humans
*Computational Biology/methods
CRISPR-Cas Systems
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
Drug Resistance, Bacterial
Phage Therapy
Bacteriophages

@article {pmid41407085,
year = {2025},
author = {Qi, H and Yang, Y and Hou, X and Chen, Y and Gong, S},
title = {Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2025.12.004},
pmid = {41407085},
issn = {1095-9130},
abstract = {CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.},
}

@article {pmid41344099,
year = {2025},
author = {Ryu, YC and Bao, G and Hwang, BH},
title = {Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {193},
number = {},
pages = {118838},
doi = {10.1016/j.biopha.2025.118838},
pmid = {41344099},
issn = {1950-6007},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; *Transfection/methods ; Plasmids/genetics ; *Gene Transfer Techniques ; *Peptides/chemistry ; Mice ; *Lipids/chemistry ; Genetic Therapy/methods ; Cell Survival ; Genetic Vectors ; },
abstract = {Efficient and safe delivery of large genetic constructs such as CRISPR/Cas9 plasmids remains a critical bottleneck in gene therapy. In this work, the peptide-assisted lipofection (PAL) system was developed as a breakthrough non-viral vector for gene delivery, specifically designed for large plasmids including CRISPR/Cas9 constructs. This system achieved exceptional transfection efficiency up to 98.7 % in HEK293T cells, surpassing conventional delivery methods such as electroporation and standard lipofections. PAL successfully delivered large plasmids up to 29 kb while maintaining high cell viability and achieved 44.1 % indel formation efficiency in gene editing experiments. Fluorescence microscopy verified PAL's efficient endosomal escape and nuclear targeting abilities. The superior performance of the PAL is attributed to its cellular uptake and endosomal escape enhanced by transfection-assisting peptide. In vivo studies in mouse models showed sustained gene expression in liver tissue, demonstrating superior performance compared to naked plasmid delivery. These results establish PAL as a versatile and promising platform for gene therapy and genome editing, offering a safer alternative to viral vectors for large genetic payload delivery.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
Animals
*Transfection/methods
Plasmids/genetics
*Gene Transfer Techniques
*Peptides/chemistry
Mice
*Lipids/chemistry
Genetic Therapy/methods
Cell Survival
Genetic Vectors

@article {pmid41329618,
year = {2025},
author = {Udemezue, VC and Shaikh, KM and Vorontsova, M and Valgepea, K},
title = {Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4967-4972},
doi = {10.1021/acssynbio.5c00456},
pmid = {41329618},
issn = {2161-5063},
mesh = {*Plasmids/genetics ; *Clostridium/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; Electroporation ; },
abstract = {Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.},
}

*Plasmids/genetics
*Clostridium/genetics/metabolism
CRISPR-Cas Systems/genetics
*Metabolic Engineering/methods
Gene Editing/methods
*Genetic Engineering/methods
Electroporation

@article {pmid41285016,
year = {2025},
author = {He, RL and Huang, BC and Wu, J and Sun, D and Li, HH and Liu, JQ and Wu, J and Liu, DF and Li, WW},
title = {One-Step Biological Upcycling of Chitin Wastes into Violacein by Engineered Chromobacterium violaceum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4733-4743},
doi = {10.1021/acssynbio.5c00472},
pmid = {41285016},
issn = {2161-5063},
mesh = {*Chromobacterium/metabolism/genetics ; *Chitin/metabolism ; *Indoles/metabolism ; *Metabolic Engineering/methods ; Fermentation ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Existing technologies for the valorization of organic wastes have been focused mainly on degradable wastes, while an efficient, low-carbon approach for the upcycling of shell waste is still lacking. Here, we report a one-step chitin biological fermentation process (CBFP), based on the construction of Chromobacterium violaceum engineered strain, for efficiently converting shell waste-derived chitin into high-value violacein. A high-efficiency CRISPR cytosine-base editor (pRK2-BE, 97% editing efficiency) was developed for C. violaceum, which demonstrated cv_4240, cv_1440, and cv_2935 as the major chitin hydrolysis genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as the major N-acetyl-glucosamine uptake pathway. The engineered strain WT/pBAD-4, co-overexpressing of cv_4240, cv_1440, cv_2935, and vioABCDE, efficiently utilized colloidal chitin and crystalline chitin as the sole carbon and nitrogen source, achieving violacein yields of 159.78 and 120.95 mg·L[-1], respectively. This study provided an economically viable and environmentally sustainable solution for green upcycling of shell waste.},
}

*Chromobacterium/metabolism/genetics
*Chitin/metabolism
*Indoles/metabolism
*Metabolic Engineering/methods
Fermentation
CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid41277095,
year = {2025},
author = {Thornbury, M and Omran, RP and Kumar, L and Knoops, A and Abushahin, R and Whiteway, M and Martin, VJJ},
title = {Tri-Functional CRISPR Screen Reveals Overexpression of QDR2 and QDR3 Transporters Increase Fumaric Acid Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4907-4922},
doi = {10.1021/acssynbio.5c00689},
pmid = {41277095},
issn = {2161-5063},
mesh = {*Fumarates/metabolism ; *Kluyveromyces/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Membrane Transport Proteins/genetics/metabolism ; },
abstract = {Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7, encoding a subunit of the mitochondrial F1F0 ATP synthase, and overexpression of QDR2 and QDR3, two previously uncharacterized members of the 12-spanner H[+] antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a ΔFUM1 background strain improved titers of fumaric acid production from 0.26 to 2.16 g L[-1]. Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus.},
}

*Fumarates/metabolism
*Kluyveromyces/metabolism/genetics
*Fungal Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Metabolic Engineering/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Membrane Transport Proteins/genetics/metabolism

@article {pmid41273852,
year = {2025},
author = {Gallucci, FP and de Camargo, JA and Viana, NI and Pimenta, RCA and Guimarães, VR and Candido, P and Leite, KRM and Nahas, WC and Dos Reis, ST},
title = {CRISPR/Cas9-mediated MMP-9 silencing inhibits bladder cancer T24 cell invasion and migration in vitro.},
journal = {Clinics (Sao Paulo, Brazil)},
volume = {80},
number = {},
pages = {100842},
pmid = {41273852},
issn = {1980-5322},
mesh = {Humans ; *Urinary Bladder Neoplasms/pathology/genetics/enzymology ; *Matrix Metalloproteinase 9/genetics/metabolism ; *Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness/genetics ; *Gene Silencing ; Apoptosis/genetics ; Blotting, Western ; Flow Cytometry ; Real-Time Polymerase Chain Reaction ; Gene Expression Regulation, Neoplastic ; },
abstract = {PURPOSE: Bladder Cancer (BCa) ranks as the tenth most common cancer worldwide, with high morbidity and mortality. Matrix Metalloproteinases (MMPs), especially MMP-9, are associated with tumor progression and metastasis. This study aimed to evaluate the effects of MMP-9 silencing using CRISPR-Cas9 in T24-luc bladder carcinoma cells.

METHODS: Guide RNAs (sgRNA1 and sgRNA2) targeting MMP-9 were cloned into pX-330 plasmids and transfected into T24-luc cells. Gene and protein expression were analyzed via RT-qPCR and Western blotting. Functional assays included flow cytometry for proliferation and apoptosis, colony formation, wound healing, and Matrigel™ invasion assays.

RESULTS: sgRNA2 significantly reduced MMP-9 gene expression, while both sgRNAs reduced protein expression. Edited cells showed decreased proliferation and colony formation, increased apoptosis, and reduced migration and invasion capacity.

CONCLUSION: CRISPR-Cas9-mediated silencing of MMP-9 inhibited cell proliferation, migration, invasion, and increased apoptosis in BCa cells, supporting that MMP-9 has an important effect on the progression of bladder cancer.},
}

Humans
*Urinary Bladder Neoplasms/pathology/genetics/enzymology
*Matrix Metalloproteinase 9/genetics/metabolism
*Cell Movement/genetics
*CRISPR-Cas Systems/genetics
Cell Proliferation/genetics
Cell Line, Tumor
Neoplasm Invasiveness/genetics
*Gene Silencing
Apoptosis/genetics
Blotting, Western
Flow Cytometry
Real-Time Polymerase Chain Reaction
Gene Expression Regulation, Neoplastic

@article {pmid41267649,
year = {2025},
author = {Matsubara, K and Hirota, M and Kajiwara, K and Senga, H and Matsui, S and Marutani, M and Seki, Y},
title = {Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {24},
pages = {},
doi = {10.1242/dev.204886},
pmid = {41267649},
issn = {1477-9129},
support = {18H02422//Japan Society for the Promotion of Science/ ; //Kwansei Gakuin University/ ; },
mesh = {Animals ; Mice ; *Transcription Factors/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Gene Expression Regulation, Developmental ; *Cell Lineage/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Humans ; Cell Differentiation/genetics ; DNA-Binding Proteins ; RNA-Binding Proteins ; },
abstract = {The network of transcription factors is dynamically reorganized during the transition from naïve- to formative-pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that POU5F1 and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation in naïve embryonic stem cells. Loss of these enhancers attenuates the upregulation of Prdm14, leading to reduced Pramel7 induction and impaired degradation of UHRF1, which consequently diminished global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in a delayed transition from the naïve to formative pluripotent state. Our findings reveal that rodent-specific enhancer insertions endow Prdm14 with a dynamic regulatory architecture, enabling both activation and repression that collectively ensure the timely exit from naïve pluripotency during early embryogenesis.},
}

Animals
Mice
*Transcription Factors/genetics/metabolism
*Enhancer Elements, Genetic/genetics
Gene Expression Regulation, Developmental
*Cell Lineage/genetics
*Pluripotent Stem Cells/metabolism/cytology
CRISPR-Cas Systems/genetics
Humans
Cell Differentiation/genetics
DNA-Binding Proteins
RNA-Binding Proteins

@article {pmid41257388,
year = {2025},
author = {Gervais, NC and Hendriks, A and Shapiro, RS},
title = {Effective Strategies for Translating CRISPR-dCas Systems to Diverse Microbes.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4624-4635},
doi = {10.1021/acssynbio.5c00537},
pmid = {41257388},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteria/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-dCas tools have widespread applications for rapidly manipulating and dissecting gene function across the microbial tree of life. However, despite their theoretical suitability for use in a broad range of species, CRISPR-dCas tools that are often initially optimized for use in model cell lines and model organisms still frequently require extensive modifications to enable their application in specific microbial organisms. Here, we review different iterations of CRISPR-dCas in microbes and the application of these techniques. We further discuss common obstacles faced and troubleshooting approaches while developing and applying CRISPR-dCas systems to a microbial organism. Finally, we suggest enhancements that can be made that may help improve the applicability of a CRISPR-dCas tool developed for nonmodel microbial organisms.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Bacteria/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41248489,
year = {2025},
author = {Sinan, S and Kooistra, RM and Rajaraman, K and Islam, Z and Madan, D and Nalefski, EA and Finkelstein, IJ},
title = {One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4714-4722},
doi = {10.1021/acssynbio.5c00463},
pmid = {41248489},
issn = {2161-5063},
mesh = {*Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Human papillomavirus 16/genetics/isolation & purification ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Papillomavirus Infections/diagnosis/virology ; DNA, Viral/genetics ; Uterine Cervical Neoplasms/virology/diagnosis ; DNA, Single-Stranded/genetics ; DNA-Directed DNA Polymerase/metabolism ; },
abstract = {CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid preamplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12a's trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in crude cell lysates of cervical cancer cells with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.},
}

*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Human papillomavirus 16/genetics/isolation & purification
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Papillomavirus Infections/diagnosis/virology
DNA, Viral/genetics
Uterine Cervical Neoplasms/virology/diagnosis
DNA, Single-Stranded/genetics
DNA-Directed DNA Polymerase/metabolism

@article {pmid41241768,
year = {2025},
author = {Lotfi, M and Farshchian Yazdi, Z and Hashemi, MR and Abbaszadegan, MR and Sharif, S},
title = {Guarding the genome: strategies for off-target detection and minimization in CRISPR/Cas9 system.},
journal = {Expert review of molecular diagnostics},
volume = {25},
number = {12},
pages = {883-902},
doi = {10.1080/14737159.2025.2581677},
pmid = {41241768},
issn = {1744-8352},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {INTRODUCTION: CRISPR-based genome editing has revolutionized molecular diagnostics and therapeutic applications, offering precise genetic modifications. However, off-target effects remain a significant challenge, potentially leading to unintended mutations and genomic instability. Addressing these concerns is crucial for ensuring the clinical safety and efficacy of CRISPR technologies.

AREAS COVERED: This review provides a comprehensive analysis of strategies for detecting and mitigating off-target effects in CRISPR genome editing. We discuss in silico, in vitro, and in vivo detection methods, highlighting their strengths and limitations. Additionally, we examine advancements in guide RNA (gRNA) engineering, improved Cas variants, optimized delivery systems, base and prime editing technologies, and anti-CRISPR proteins as key approaches to enhance genome-editing precision.

EXPERT OPINION: While significant progress has been made in off-target detection and mitigation, challenges remain in accurately assessing unintended genomic modifications in vivo and ensuring long-term safety in clinical applications. Future research should focus on developing high-fidelity CRISPR variants, refining computational prediction models, and integrating multi-modal detection systems. Advancing delivery mechanisms and regulatory strategies will be essential for translating CRISPR into safe and effective therapeutic interventions.},
}

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