@article {pmid39976035,
year = {2026},
author = {Sen, A and Singh, V and Dwivedi, S and Agrawal, R and Bansal, A and Shekhar, S and Kushwaha, SS},
title = {Gene Therapy and Gene Editing: Current Trends and Future Prospects of Molecular Medicine.},
journal = {Current gene therapy},
volume = {26},
number = {1},
pages = {53-66},
pmid = {39976035},
issn = {1875-5631},
mesh = {Humans ; *Genetic Therapy/trends/methods ; *Gene Editing/trends/methods/ethics ; CRISPR-Cas Systems ; *Molecular Medicine/trends/methods ; Precision Medicine ; *Genetic Diseases, Inborn/therapy/genetics ; Neoplasms/therapy/genetics ; },
abstract = {Gene therapy and genome editing have emerged as transformative approaches in the management of a diverse range of genetic and acquired diseases. This evaluation offers a thorough examination of the present state and prospects of these innovative technologies. Gene therapy is a prospective approach to the treatment and prevention of a variety of conditions, including complex cancers and inherited genetic disorders, which entail the introduction, removal, or modification of genetic material within a patient's cells. Genome editing, particularly through techniques such as CRISPR-Cas9, enables targeted corrections of genetic defects and opens new possibilities for personalized medicine by allowing for precise modifications at the DNA level. The review addresses the ethical implications, clinical applications, and significant advancements of these technologies. This article endeavors to underscore the substantial influence of gene therapy and genome editing on contemporary medicine by assessing the most recent research and clinical trials, thereby emphasizing their potential to revolutionize disease treatment and management.},
}

Humans
*Genetic Therapy/trends/methods
*Gene Editing/trends/methods/ethics
CRISPR-Cas Systems
*Molecular Medicine/trends/methods
Precision Medicine
*Genetic Diseases, Inborn/therapy/genetics
Neoplasms/therapy/genetics

@article {pmid40129147,
year = {2026},
author = {Khan, Z and Mumtaz, and Gupta, S and Mehan, S and Sharma, T and Kumar, M and Maurya, PK and Sharma, AK and Gupta, GD and Narula, AS},
title = {CRISPR-Cas9: Transforming Functional Genomics, Precision Medicine, and Drug Development - Opportunities, Challenges, and Future Directions.},
journal = {Current gene therapy},
volume = {26},
number = {1},
pages = {160-172},
pmid = {40129147},
issn = {1875-5631},
support = {CRG/2021/001009//Department of Science and Technology, Science and Engineering Board, DSTSERB/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Genomics/methods ; *Drug Development ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a groundbreaking gene-editing technology that enables scientists to make precise changes to the DNA of living organisms. It was first discovered in Escherichia coli and emerged as a breakthrough tool in molecular biology. This technique is essential because of its adaptability, affordability, and ease of use. It uses the adaptive immune response of bacteria and archaea to repel viral invasions. It significantly influences drug discovery, functional genomics, disease models, and pharmaceutical research. CRISPR-Cas9 is a better and more accurate way to change genes than other methods, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). This technology promotes the generation of double-strand breaks in DNA, allowing for precise genetic alterations required for therapeutic target identification and confirmation. Functional genomics enables high-throughput screening (HTS) to identify gene functions, disease causes, and therapeutic targets. CRISPR-Cas9 increases drug development by enabling Cas9 to create novel antimicrobial drugs and cancer therapies. It has also helped to generate disease models, advance our understanding of neurodegenerative and other diseases, test a variety of chemicals, and facilitate precise genetic changes. Despite its promise, ethical considerations and the possibility of off-target effects require careful evaluation to ensure its safe and effective clinical application. This study investigates the current and future possibilities of CRISPR-Cas9 in drug development, focusing on its transformational influence and addressing the challenges and limitations of its therapeutic application.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
*Genomics/methods
*Drug Development
*Precision Medicine/methods
*Genetic Therapy/methods
Animals

@article {pmid41427901,
year = {2026},
author = {Matsumoto, K and Yamamoto, W and Fukutomi, Y and Koshikawa, S},
title = {Functions of melanin synthesis genes, yellow and tan, in wing pigmentation revealed by CRISPR/Cas9-mediated mutagenesis in Drosophila guttifera.},
journal = {Insect molecular biology},
volume = {35},
number = {3},
pages = {246-256},
doi = {10.1111/imb.70024},
pmid = {41427901},
issn = {1365-2583},
support = {//Japan Society for the Promotion of Science/ ; JP17K19427//KAKENHI/ ; JP24K21982//KAKENHI/ ; JP25K02019//KAKENHI/ ; },
mesh = {Animals ; *Pigmentation/genetics ; CRISPR-Cas Systems ; *Melanins/biosynthesis/genetics ; Wings, Animal/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/metabolism/physiology ; Mutagenesis ; },
abstract = {Colour pattern formation is a key model for studying evolutionary and developmental mechanisms. In the fruit fly Drosophila guttifera, which exhibits distinctive polka-dot wing pigmentation, we investigated the roles of two putative melanin synthesis genes, yellow and tan, using CRISPR/Cas9-mediated genome editing. We established multiple mutant strains with lesions in either gene and found that both genes were essential for normal pigmentation intensity in wing spots, though the patterns themselves persisted. Double mutants showed further reduction in pigmentation, indicating additive effects but not complete loss of patterning. Ectopic expression of wingless failed to induce normal pigmentation in yellow or tan mutants, demonstrating that both genes act downstream of wingless and are required for its pigmentation-inducing function. Furthermore, mosaic phenotypes in G0 individuals revealed quasi-cell-autonomous functions of tan, suggesting that pigmentation in D. guttifera wings depends on local availability of precursors rather than solely on transport via wing veins. This study establishes D. guttifera as a genetically tractable system for functional analyses and contributes to understanding the molecular basis of insect colour pattern formation.},
}

Animals
*Pigmentation/genetics
CRISPR-Cas Systems
*Melanins/biosynthesis/genetics
Wings, Animal/metabolism
*Drosophila Proteins/genetics/metabolism
*Drosophila/genetics/metabolism/physiology
Mutagenesis

@article {pmid41844662,
year = {2026},
author = {Zhang, J and Wang, Q and Cheng, Z and Liu, J and Liu, Q and Qi, S and Chen, Z and Duan, Y and Liu, Z and Jia, J and Li, C},
title = {Enhancer-mediated Etv4 activation stimulates osteogenic differentiation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41844662},
issn = {2041-1723},
support = {82072499, 32270610//National Natural Science Foundation of China (National Science Foundation of China)/ ; L242120//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {Animals ; *Osteogenesis/genetics ; Mice ; *Cell Differentiation/genetics ; Osteoblasts/metabolism/cytology ; *Enhancer Elements, Genetic/genetics ; STAT3 Transcription Factor/metabolism ; *Proto-Oncogene Proteins c-ets/genetics/metabolism ; Cell Line ; Humans ; Mice, Knockout ; CRISPR-Cas Systems ; Osteoporosis/genetics ; *Adenovirus E1A Proteins/genetics/metabolism ; },
abstract = {Enhancers, as cis-regulatory elements, play pivotal roles in transcriptional homeostasis. The abnormality in enhancers is highly associated with various diseases, including osteoporosis. However, the landscape of active enhancers underlying bone diseases remains incomplete. By conducting an integrative analysis of transcriptome and ChIP-seq data, we identify enh11 as an active enhancer during osteoblastogenesis. CRISPR/Cas9-mediated deletion of enh11 inhibits cell differentiation of pre-osteoblast MC3T3-E1 cells. The osteoblast-specific knockout of enh11 reduces bone formation and decreases bone mass in mice. In addition, Etv4 is identified as the downstream target of enh11. Functional experiments both in vitro and in vivo validate that Etv4 promotes osteogenesis and bone formation. Mechanistically, enh11 upregulates the expression of Etv4 to promote osteogenesis, probably via binding to the transcription factor Stat3. These findings not only deepen our comprehension of the molecular mechanisms of enh11 underlying bone formation but also highlight enh11 and Etv4 as promising therapeutic targets for osteoporosis.},
}

Animals
*Osteogenesis/genetics
Mice
*Cell Differentiation/genetics
Osteoblasts/metabolism/cytology
*Enhancer Elements, Genetic/genetics
STAT3 Transcription Factor/metabolism
*Proto-Oncogene Proteins c-ets/genetics/metabolism
Cell Line
Humans
Mice, Knockout
CRISPR-Cas Systems
Osteoporosis/genetics
*Adenovirus E1A Proteins/genetics/metabolism

@article {pmid41882696,
year = {2026},
author = {Ren, W and Yang, M and Zhou, Y and Yang, Y and Li, H and Chen, Y and Li, S and Pang, Y},
title = {An ultra-sensitive cell-free DNA-based diagnostic assay for Tuberculous pleurisy utilizing the CRISPR-Cas13a system.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {25},
number = {1},
pages = {},
pmid = {41882696},
issn = {1476-0711},
support = {S2023050//Popularization and application of appropriate medical and health technology in Guangxi Province/ ; 20230484295//Beijing Nova Program/ ; KJ2024CX028//Beijing Tongzhou District Science and Technology Commission/ ; 2024-4-1042//Health Improvement and Research/ ; },
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Cell-Free Nucleic Acids/analysis ; CRISPR-Cas Systems ; *DNA, Bacterial/analysis ; *Molecular Diagnostic Techniques/methods/statistics & numerical data ; *Mycobacterium tuberculosis/isolation & purification ; Pleural Effusion/microbiology ; Prospective Studies ; Sensitivity and Specificity ; *Tuberculosis, Pleural/diagnosis ; },
abstract = {BACKGROUND: Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free Mycobacterium tuberculosis (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.

METHODS: Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).

RESULTS: Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, P < 0.001) and Xpert (42.9%, P < 0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, P < 0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both P < 0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.

CONCLUSIONS: The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.},
}

Adolescent
Adult
Aged
Aged, 80 and over
Female
Humans
Male
Middle Aged
Young Adult
Cell-Free Nucleic Acids/analysis
CRISPR-Cas Systems
*DNA, Bacterial/analysis
*Molecular Diagnostic Techniques/methods/statistics & numerical data
*Mycobacterium tuberculosis/isolation & purification
Pleural Effusion/microbiology
Prospective Studies
Sensitivity and Specificity
*Tuberculosis, Pleural/diagnosis

@article {pmid41963468,
year = {2026},
author = {Park, SB and Kim, JS and Ha, Y and Kim, MS and Kim, TW},
title = {Human pluripotent stem cell engineering with CRISPR-Cas9 for Parkinson's disease.},
journal = {Experimental & molecular medicine},
volume = {58},
number = {4},
pages = {993-1009},
pmid = {41963468},
issn = {2092-6413},
support = {RS-2024-00351442//National Research Foundation of Korea (NRF)/ ; RS-2025-25437095//National Research Foundation of Korea (NRF)/ ; },
mesh = {Humans ; *Parkinson Disease/therapy/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; Dopaminergic Neurons/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Animals ; Gene Editing/methods ; Cell Differentiation ; *Cell Engineering/methods ; },
abstract = {Parkinson's disease (PD) entails loss of substantia nigra dopamine (DA) neurons and α-synuclein pathology. Currently, no effective disease-modifying therapies have been developed. Human pluripotent stem cells (hPS cells) can generate DA neurons on scale, enabling human genetic PD modeling of mitochondrial, lysosomal and synaptic connection failure that leads to DA neuron degeneration. Clustered regularly interspaced short palindromic repeats (CRISPR) extends this human model by providing causal, isogenic interrogation and transcriptional regulation of PD genes and reporter knock-ins that support purification and high-content screening. hPS cell-based DA cell grafts can restore motor function yet face >90% acute cell death and product heterogeneity in vivo post implantation. CRISPR enabled not only an in vivo cell survival screen to identify the cell death regulators but also a reporter-guided enrichment of DA neurons and chemogenetic control of grafted DA cell function in vivo. Here we summarize this progress and outline a practical road map to accelerate the development of precise human models and advanced hPS cell-based cell therapies for PD.},
}

Humans
*Parkinson Disease/therapy/genetics/metabolism/pathology
*CRISPR-Cas Systems
Dopaminergic Neurons/metabolism
*Pluripotent Stem Cells/metabolism/cytology
Animals
Gene Editing/methods
Cell Differentiation
*Cell Engineering/methods

@article {pmid42003707,
year = {2026},
author = {Wu, Z and Jin, F and Zhu, W and Zhong, W and Qi, T and Luo, S and Liu, Q and Cai, Z and Dai, C and Chai, Z and He, Y and Rui, Y and Miao, Y and Zheng, L and Fu, Q},
title = {Photoactivated Digital Recombinase Polymerase Amplification/CRISPR-Cas12a Assay for Point-of-Care of BK Polyomavirus Quantification.},
journal = {ACS nano},
volume = {20},
number = {17},
pages = {13301-13313},
doi = {10.1021/acsnano.6c02651},
pmid = {42003707},
issn = {1936-086X},
mesh = {*BK Virus/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; DNA, Viral/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BK polyomavirus (BKV) serves as a critical biomarker for optimizing immunosuppressive therapy and preventing graft failure in kidney transplant recipients. Quantitative PCR (qPCR), the current gold standard for BKV load quantification, relies on batch-specific standard curves. For kidney transplant recipients, this not only elevates the risk of cross-contamination but also entails considerable economic burdens. Therefore, developing BKV quantification technologies independent of batch-specific standard curves is of great clinical significance for this patient population. The combination of CRISPR-Cas12a with recombinase polymerase amplification (RPA), termed DETECTR, offers notable advantages for digital nucleic acid analysis. However, due to the high viscosity of RPA reagents, the generation of high-throughput, uniform RPA microdroplets remains a significant technical challenge. In the present study, we developed a centrifugal RPA microdroplet generation method based on commercial capillaries, facilitating the production of high-throughput, uniform RPA microdroplets (23.1 μm in diameter) via simple centrifugation. Furthermore, by integrating a light-controlled RPA-CRISPR-Cas12a system, we established photoactivated digital DETECTR (pd-DETECTR) for precise, point-of-care, and cost-effective BKV quantification. When combined with a smartphone-based reader, the pd-DETECTR assay can be completed within 42 min. Clinical validation demonstrated a strong correlation (R[2] = 0.9801) with qPCR results, exhibiting high sensitivity (100.0%), specificity (98.0%), and accuracy (99.0%). The pd-DETECTR provides a rapid, convenient, and cost-effective tool for BKV load analysis, which can significantly reduce the economic burden and risk of opportunistic infections in kidney transplant recipients, thus holding significant clinical value.},
}

*BK Virus/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
*Recombinases/metabolism/genetics
*Nucleic Acid Amplification Techniques/methods
*Point-of-Care Systems
DNA, Viral/genetics/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42008698,
year = {2026},
author = {Wang, H and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/dCas9-Assisted On-Bead Multiplex Detection (BeadPlex2) for Genetically Modified Crops.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12586-12595},
doi = {10.1021/acs.analchem.5c08192},
pmid = {42008698},
issn = {1520-6882},
mesh = {*Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; Glycine max/genetics ; Zea mays/genetics ; Spectrum Analysis, Raman ; *Crops, Agricultural/genetics ; Benzothiazoles ; },
abstract = {This study leverages the precise recognition ability of CRISPR/dCas9 and the Raman coding feature of the gap-enhanced Raman tag-encoded magnetic beads (MagGERTs) to create a unique on-bead nucleic acid detection platform (BeadPlex2) for accurate and multiplex nucleic acid detection, which was proven to be applicable for the identification of diverse genetically modified (GM) events. Five distinct MagGERTs (MB@Au[Ra]) encoded with different Raman reporters (Ras) were constructed, followed by the conjugation of dCas9/single guide RNA (sgRNA) complexes in which the sgRNAs were explicitly designed for different target genes of GM events. These coding units could recognize and capture target double-stranded nucleic acid (dsDNA) sequences by the dCas9/sgRNA complexes. Then, SYBR Green I was applied to highlight positive beads by binding to target dsDNA due to its fluorescent emission under an imaging system. Decoding Raman signals from the Ras of the MagGERTs achieved the high-specific identification of GM events. Our BeadPlex2 platform has been demonstrated to be applicable for detecting GM maize and soybean seeds with high accuracy comparable to qPCR. This platform opens a new way to detect multiple target nucleic acids simultaneously and offers a powerful strategy for identifying genetically modified organisms.},
}

*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems/genetics
Glycine max/genetics
Zea mays/genetics
Spectrum Analysis, Raman
*Crops, Agricultural/genetics
Benzothiazoles

@article {pmid42011754,
year = {2026},
author = {Yang, G and Fang, Y and Liu, Y and Deng, Y and Nie, L and Li, Z and Li, S and Chen, Z and Su, E and Zai, Y and Umar Siddiqui, AM and He, N},
title = {Rapid and Specific Detection of Gastric Cancer EVs Using a Cas12a-Powered Aptasensor with a Novel Targeting Aptamer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12529-12541},
doi = {10.1021/acs.analchem.5c08039},
pmid = {42011754},
issn = {1520-6882},
mesh = {*Stomach Neoplasms/diagnosis/pathology/metabolism ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; SELEX Aptamer Technique ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Cell Line, Tumor ; CRISPR-Cas Systems ; Biomarkers, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Gastric cancer remains a predominant contributor to global cancer-related mortality, characterized by a pronounced disparity in five-year survival rates between early stage (>90%) and advanced-stage (<30%) disease. This disparity underscores the urgent necessity for accessible early detection methods. Present diagnostic approaches, such as serum biomarkers and endoscopy, either lack adequate sensitivity or are invasive. Extracellular vesicles (EVs) represent promising biomarkers for liquid biopsy; however, a major limitation is the lack of probes that can specifically identify EVs derived from gastric cancer, as most existing markers are broad-spectrum and exhibit low specificity. To address this limitation, we isolated high-purity EVs from gastric cancer cells and utilized a combined immunomagnetic bead-based SELEX strategy to identify a novel aptamer, H-EV-4-1, which demonstrates high affinity (Kd = 13.32 ± 2.69 nM) and specificity for gastric cancer EVs. Subsequently, this aptamer was incorporated into a CRISPR-Cas12a-based biosensor. The aptamer was hybridized with a biotinylated oligonucleotide (H1-biotin) and immobilized on magnetic beads. Upon binding of the target EVs, the aptamer was displaced, thereby exposing H1-biotin to activate the Cas12a/crRNA complex. This activation induced the trans-cleavage of a fluorescent reporter, producing a quantifiable signal. This aptasensor facilitates the rapid, highly sensitive, and specific detection of gastric cancer EVs, presenting a promising platform for the development of noninvasive, point-of-care early diagnostic tools.},
}

*Stomach Neoplasms/diagnosis/pathology/metabolism
*Aptamers, Nucleotide/chemistry/metabolism
Humans
*Extracellular Vesicles/metabolism/chemistry
SELEX Aptamer Technique
*CRISPR-Associated Proteins/metabolism/genetics
*Biosensing Techniques/methods
Cell Line, Tumor
CRISPR-Cas Systems
Biomarkers, Tumor
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid42024570,
year = {2026},
author = {Liu, X and Shi, F and Luo, G and Wang, Q and Deng, F and Luo, X and Huo, D and Hou, C},
title = {A Disposable CRISPR-Nanozyme Electrochemical Biosensor for Rapid and Sensitive Detection of Breast Cancer Circulating Tumor DNA.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12802-12810},
doi = {10.1021/acs.analchem.6c00173},
pmid = {42024570},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Circulating Tumor DNA/analysis/blood/genetics ; *Breast Neoplasms/blood/genetics/diagnosis ; *Electrochemical Techniques/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Female ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) serves as a promising next-generation biomarker for noninvasive cancer screening and monitoring. In this work, we report an electrochemical biosensor for ctDNA detection. The sensor is constructed based on the synergistic integration of CRISPR/Cas12a and PB-Au NPs. CRISPR/Cas12a provides precise target recognition and triggers trans-cleavage, while the nanozyme enables strong signal amplification through its catalytic activity. Using a disposable carbon-fiber paper as the biosensing interface, we developed this sensitive detection strategy. On this interface, a PB-AuNP-labeled single-stranded DNA reporter is immobilized. In the presence of target ctDNA, activated Cas12a cleaves the reporter, releasing the nanozyme and resulting in a quantifiable decrease in the TMB oxidation current. This dual-amplification strategy achieves a detection limit of 860 aM (S/N = 3) with a linear range from 1 fM to 1 nM. Overall, this approach provides a satisfactory demonstration toward the realization of a low-cost and highly sensitive biosensor for ctDNA detection.},
}

*Biosensing Techniques/methods
Humans
*Circulating Tumor DNA/analysis/blood/genetics
*Breast Neoplasms/blood/genetics/diagnosis
*Electrochemical Techniques/methods
Gold/chemistry
Metal Nanoparticles/chemistry
Female
*CRISPR-Cas Systems
Limit of Detection

@article {pmid42027134,
year = {2026},
author = {Xu, L and Zhao, X and Meng, X and Chen, J and Chen, P},
title = {Multiselective Recognition of Metal Ion-Nucleic Acid Complexes by CRISPR/Cas12a and Quantum Dots Enables the Profiling of Circulating Tumor DNA in Breast Cancer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {13120-13133},
doi = {10.1021/acs.analchem.6c01333},
pmid = {42027134},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *Breast Neoplasms/genetics/blood/diagnosis ; Humans ; *CRISPR-Cas Systems ; Female ; *Circulating Tumor DNA/blood/genetics ; *Silver/chemistry ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; },
abstract = {The rapid, noninvasive detection of circulating tumor DNA (ctDNA) is vital for the diagnosis and staging of breast cancer (BC). In this study, we developed a homogeneous CRISPR/Cas12a fluorescent platform using a hierarchical grape-cluster rolling circle amplification (GCRCA) nanomaterial to detect the PIK3CA E542K mutation. A pivotal discovery of this study is that activated Cas12a efficiently cleaves metal ion-mediated cytosine-Ag[+]-cytosine base pairs, which enables direct coupling between enzymatic activity and signal transduction. GCRCA, assembled by precise hybridization of long-chain RCA concatemers with auxiliary circular DNA, features Ag[+]-bridged dual-ring units that sequester both target sequences and Ag[+] reporters within a self-shielding framework. Upon target recognition, activated Cas12a dismantles the GCRCA architecture, initiating an autocatalytic feedback loop that releases caged Ag[+] to quench the quantum dot fluorescence. This label-free assay achieved attomolar sensitivity within 30 min without enzymatic preamplification or complex nucleic acid extraction. Importantly, the platform exhibits excellent sequence selectivity, enabling precise discrimination of single-base mutations against closely related sequences. Validation of 42 clinical plasma samples achieved 100% diagnostic specificity for BC. For staging, the platform yielded a sensitivity of 100%, a specificity of 92.3%, and an area under the curve of 0.978. With its exceptional sensitivity and operational simplicity, this platform offers a promising approach for precise ctDNA-based BC detection and staging, demonstrating significant translational clinical potential.},
}

*Quantum Dots/chemistry
*Breast Neoplasms/genetics/blood/diagnosis
Humans
*CRISPR-Cas Systems
Female
*Circulating Tumor DNA/blood/genetics
*Silver/chemistry
Class I Phosphatidylinositol 3-Kinases/genetics
*Endodeoxyribonucleases/metabolism
*CRISPR-Associated Proteins/metabolism
Nucleic Acid Amplification Techniques
Bacterial Proteins

@article {pmid42083602,
year = {2025},
author = {Taebi, S and Eskandari, F and Kohandani, M and Manoochehrabadi, T and Nasiri, H},
title = {Genetic Engineering in Hematopoietic Stem Cells for β-Hemoglobinopathies Treatment: Advances, Challenges, and Clinical Translation.},
journal = {International journal of hematology-oncology and stem cell research},
volume = {19},
number = {4},
pages = {399-423},
pmid = {42083602},
issn = {2008-3009},
abstract = {β-hemoglobinopathies rank among the most prevalent inherited blood disorders globally. Traditional management strategies are primarily palliative and often associated with significant challenges, including iron overload and limited long-term efficacy. Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for transfusion-dependent patients, but its broader applicability is constrained by factors that limit its use. Utilizing viral vectors and gene-editing tools, particularly CRISPR-Cas9 technology, researchers have developed therapies that target the root causes of these disorders. These innovative approaches have demonstrated substantial therapeutic potential, accompanied by favorable safety profiles, in clinical settings. Since the initial investigations, the genome editing tool has rapidly advanced for genetic abnormalities, particularly monogenic blood diseases, including β-hemoglobinopathies. This method suggests an approach with lower concerns in viral gene integration and insertional mutagenesis issues. This review comprehensively surveys the therapeutic strategies for β-thalassemia and sickle cell disease (SCD) currently in preclinical and clinical development, with a focus on the evolving treatment paradigm. Looking forward, critical research priorities include optimizing the efficiency and specificity of gene-editing platforms and pioneering novel delivery systems to guarantee both therapeutic efficacy and clinical safety.},
}

@article {pmid42084692,
year = {2026},
author = {Minami, A and Shimizu, M and Tamaki, S and Nishinarizki, V and Yosua, and Mochida, K},
title = {Affordable CRISPR RNP-Based Genome Editing in Euglena gracilis.},
journal = {Current protocols},
volume = {6},
number = {5},
pages = {e70357},
doi = {10.1002/cpz1.70357},
pmid = {42084692},
issn = {2691-1299},
support = {JPMJSA2204//This work was supported by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) through the Science and Technology Research Partnership for Sustainable Development (SATREPS) Program/ ; },
mesh = {*Gene Editing/methods/economics ; *Euglena gracilis/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.},
}

*Gene Editing/methods/economics
*Euglena gracilis/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42084860,
year = {2026},
author = {Tian, Y and Li, C and Zhao, E and Chen, Y and Shen, X and Xu, S and Yu, Y and Sun, L},
title = {Recent Advances in the Detection of Plant Diseases Based on the CRISPR-Cas System.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07871},
pmid = {42084860},
issn = {1520-6882},
}

@article {pmid41188232,
year = {2025},
author = {Fu, BXH and Xu, A and Li, H and Johnson, DE and Grandis, JR and Gilbert, LA},
title = {Loss of Fanconi anemia proteins causes a reliance on lysosomal exocytosis.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {791},
pmid = {41188232},
issn = {2041-4889},
mesh = {Humans ; Cell Line ; *Exocytosis ; *Lysosomes/metabolism ; *Fanconi Anemia/genetics/metabolism/pathology ; Autophagy ; CRISPR-Cas Systems ; Genome, Human ; DNA Repair ; Neoplasms/genetics ; },
abstract = {Mutations in the Fanconi Anemia (FA) pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatments for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA pathway function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied by decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures in vitro across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.},
}

Humans
Cell Line
*Exocytosis
*Lysosomes/metabolism
*Fanconi Anemia/genetics/metabolism/pathology
Autophagy
CRISPR-Cas Systems
Genome, Human
DNA Repair
Neoplasms/genetics

@article {pmid41400455,
year = {2026},
author = {Mukherjee, S and Kumar, M},
title = {CRISPR: a precise genome editing strategy for the treatment of hepatocellular carcinoma.},
journal = {Expert review of anticancer therapy},
volume = {26},
number = {5},
pages = {599-614},
doi = {10.1080/14737140.2025.2606090},
pmid = {41400455},
issn = {1744-8328},
mesh = {Humans ; *Carcinoma, Hepatocellular/therapy/genetics/pathology ; *Liver Neoplasms/therapy/genetics/pathology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; Prognosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Genetic Therapy/methods ; Receptors, Chimeric Antigen ; },
abstract = {INTRODUCTION: The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing tool provides novel therapeutic alternatives by promoting the gene alteration in adaptive T cells or malignant cells to combat Hepatocellular Carcinoma (HCC). More successful cancer treatments are now possible due to the capacity of precisely locating and modifying particular genetic abnormalities that promote malignancy growth and metastasis.

AREAS COVERED: In this review, we address ongoing clinical trials, the possible similarities between CRISPR-based cancer treatments and current therapeutic choices, and how CRISPR technology can improve treatment outcomes for HCC while using the latest safety measures. Additionally, this analysis sheds light on the existing obstacles and potential future possibilities of applying CRISPR technology to the management of HCC, with a final objective of enhancing patient results and completely changing the field of HCC therapies.

EXPERT OPINION: The urgent need for innovative therapies is underscored by the poor prognosis associated with severe hepatocellular carcinoma, despite recent advancements in clinical therapies. Through a special emphasis on invivo cancer cell targeting along with the generation of chimeric antigen receptor (CAR) T cells, including T cell receptor (TCR) T cells, this review analyses the uses of CRISPR methods in the therapy of HCC.},
}

Humans
*Carcinoma, Hepatocellular/therapy/genetics/pathology
*Liver Neoplasms/therapy/genetics/pathology
*Gene Editing/methods
CRISPR-Cas Systems
Animals
Prognosis
Clustered Regularly Interspaced Short Palindromic Repeats
Immunotherapy, Adoptive/methods
T-Lymphocytes/immunology
Genetic Therapy/methods
Receptors, Chimeric Antigen

@article {pmid41690142,
year = {2026},
author = {Huang, Z and Ding, Y and Xu, H and Zhang, Q and Wang, X and Ding, H and Zhang, J and Wang, Y and Wang, Y and Jiao, P},
title = {Development of an RT-ERA-CRISPR/Cas12a assay for duck Tembusu virus.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106612},
pmid = {41690142},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Flavivirus/isolation & purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Flavivirus Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; China ; },
abstract = {Duck Tembusu virus (DTMUV) has continued to threaten the duck industry in China since 2010. Therefore, the establishment of a rapid, specific, and sensitive method for the field detection of DTMUV is urgently needed. Herein, a reverse transcription enzymatic recombinase amplification (RT-ERA) assay was combined with the CRISPR/Cas12a system to target the DTMUV C gene. This assay exhibited high specificity, effectively distinguishing DTMUV from other common avian viruses. Its limit of detection reached 1 copy/μL DTMUV RNA. Moreover, this assay can be completed at 42 °C within 15 min using a thermostatic water bath. Additionally, we tested 30 clinical samples from infected ducks using this assay, and the results showed 100% concordance with SYBR green quantitative PCR results. In summary, this rapid, specific, and sensitive assay shows promising potential for DTMUV detection in the field.},
}

Animals
*Ducks
*Flavivirus/isolation & purification/genetics
*Poultry Diseases/virology/diagnosis
*Flavivirus Infections/veterinary/diagnosis/virology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/veterinary/methods
China

@article {pmid41762975,
year = {2026},
author = {Guo, L and Cui, K and Yang, Y and Dong, S and Chen, Y and Liu, K and Lei, X and Duan, B and Zhao, Y and Lv, X and Bai, R and Zheng, M},
title = {Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106681},
pmid = {41762975},
issn = {1525-3171},
mesh = {Animals ; *Eimeria/isolation & purification/classification/genetics ; *Chickens ; *Coccidiosis/veterinary/diagnosis/parasitology ; *Poultry Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Feces/parasitology ; Reproducibility of Results ; },
abstract = {Chicken coccidiosis, caused by infection with one or more of the seven Eimeria spp., is a major challenge in global poultry production. Rapid and accurate identification at the species level is critical for guiding targeted treatment strategies, minimizing antibiotic misuse, and mitigating disease transmission. In this study, we developed a point-of-care testing (POCT) platform, E-MRC12a (Eimeria-Multiplex RAA-CRISPR/Cas12a), which integrates multiplex recombinase-aided amplification (RAA) with CRISPR/Cas12a technology for the simultaneous detection of all seven Eimeria species in chicken fecal samples. Key assay parameters were optimized to balance detection performance and operational cost. The system was comprehensively evaluated for its sensitivity, specificity, reproducibility, and field applicability. E-MRC12a enables visual, one-pot detection of as few as 1 oocyst/μL. The process from sample loading to result interpretation required 1 h, while the total time from initial sample processing to final result readout was approximately 2 h. The assay exhibited high specificity with no cross-reactivity among Eimeria species, and demonstrated 100% concordance with conventional diagnostic methods in clinical validation. This rapid, field-deployable platform provides a species-specific coccidiosis diagnostic solution, supporting epidemiological surveillance and multivalent anticoccidial vaccine development.},
}

Animals
*Eimeria/isolation & purification/classification/genetics
*Chickens
*Coccidiosis/veterinary/diagnosis/parasitology
*Poultry Diseases/diagnosis/parasitology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Feces/parasitology
Reproducibility of Results

@article {pmid41813589,
year = {2026},
author = {Yu, T and Gao, P and Wang, K and Chen, B and Chu, Z and Liu, S and Hong, J and Zhang, X and Zhang, Z and Lv, R and Liu, B and Xun, H},
title = {Loss-of-Function Mutation in TaZIP4-B2 and TaMSH7-3D Fuels Karyotypic Variation, Phenotypic Diversity, and Enables Rapid Evolution of Tolerance to Salinity Stress.},
journal = {Plant, cell & environment},
volume = {49},
number = {6},
pages = {3455-3469},
doi = {10.1111/pce.70487},
pmid = {41813589},
issn = {1365-3040},
support = {2022YFF1003303//National Key Research and Development Programme of China/ ; 32572354//Natural Science Foundation of China/ ; 32061143001//Israel Science Foundation (ISF)-China National Natural Science Foundation (NSFC)/ ; },
mesh = {*Triticum/genetics/physiology ; Phenotype ; *Loss of Function Mutation/genetics ; Karyotype ; *Plant Proteins/genetics/metabolism ; *Salt Stress/genetics ; *Salt Tolerance/genetics ; Meiosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Allopolyploid species often contain specific genes dedicated to suppressing meiotic homoeologous pairing. In common wheat, TaZIP4-B2 and TaMSH7-3D fulfil this role. Nevertheless, to what extent the loss-of-function of these genes may lead to meiotic breakdown in wheat itself and hence generate karyotypic heterogeneity remains incompletely understood. Here, we show that CRISPR/Cas9-generated loss-of-function mutation of either or both TaZIP4-B2 and TaMSH7-3D leads to disrupted meiosis, triggering widespread karyotypic instability including both numerical and structural chromosomal variations (NCVs and SCVs). NCVs predominantly occurred in the D subgenome, involving preferential gains of 2A/4B/5D and losses of 6A/5B/2D, while frequencies of SCVs among subgenomes followed the order of subgenomes D > A > B, with 6A/5B/2D showing the most rearrangements. Notably, karyotypic variation in Tazip4-B2/Tamsh7-3D double mutants showed initial rapid accumulation followed by gradual stabilization across generations. Karyotypic heterogeneity caused extensive phenotypic diversity, including several key agronomic traits. Notably, Tazip4-B2/Tamsh7-3D double mutant showed more intercalary insertional translocations than the classical ph1b deletion mutant, suggesting its advantage in alien genetic introgression. Moreover, tolerance to strong salinity emerged in progenies of the mutants due to karyotypic variation. Our findings demonstrate that the loss-of-function mutation of TaZIP4-B2/TaMSH7-3D promotes rapid karyotype variability, phenotypic diversity, and environmental adaptability in wheat itself, suggesting a novel possibility for wheat improvement by karyotypic renovation.},
}

*Triticum/genetics/physiology
Phenotype
*Loss of Function Mutation/genetics
Karyotype
*Plant Proteins/genetics/metabolism
*Salt Stress/genetics
*Salt Tolerance/genetics
Meiosis/genetics
CRISPR-Cas Systems

@article {pmid41874550,
year = {2026},
author = {Hsu, JY and Lu, WH and Chang, YY and Chiang, PC and King, PC and Hsu, YH and Huang, YS},
title = {Generation of Ucp1-ires-Cre knock-in mice to enhance specificity and efficiency of gene targeting in brown adipose tissue.},
journal = {American journal of physiology. Endocrinology and metabolism},
volume = {330},
number = {5},
pages = {E675-E683},
doi = {10.1152/ajpendo.00533.2025},
pmid = {41874550},
issn = {1522-1555},
support = {NHRI-EX109-10719SI//National Health Research Institutes (NHRI)/ ; NSTC111-2311-B-001-020-MY3//National Science and Technology Council (NSTC)/ ; AS-GC-111-L04//Academia Sinica (AS)/ ; },
mesh = {Animals ; *Uncoupling Protein 1/genetics/metabolism ; *Adipose Tissue, Brown/metabolism ; Mice ; *Integrases/genetics/metabolism ; Mice, Transgenic ; *Gene Knock-In Techniques/methods ; Thermogenesis/genetics ; Male ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Gene Targeting/methods ; Female ; },
abstract = {Brown adipose tissue (BAT) secretes cytokines that influence the function of other tissues. Given the widespread distribution of brown fat depots, we generated BAT-lacking (ΔBAT) mouse models by specifically eliminating brown adipocytes using the Cre-loxP system combined with a floxed-stop diphtheria toxin A (DTA) cassette. Uncoupling protein 1 (UCP1) is essential for BAT thermogenesis and exhibits a highly restricted expression pattern, so it was chosen to direct BAT-specific Cre recombinase expression. We used CRISPR-Cas9 to insert an ires-Cre sequence downstream of the UCP1 stop codon, developing the novel knock-in line, Ucp1-Cre[YH]. Ucp1-Cre[YH] and transgenic line TgUcp1-Cre[Evdr] mouse (Ucp1-Cre[Evdr]) were crossed with Ai14-tdTomato and floxed-CPEB2 mice to assess Cre specificity and efficiency. ΔBAT mice were then generated by crossing each Cre line with floxed-stop DTA mice, followed by assessments of locomotor activity, body weight, and glucose tolerance. Although both Cre lines showed cold-enhanced expression, Ucp1-Cre[Evdr] exhibited considerably lower Cre levels in BAT compared with Ucp1-Cre[YH] mice, leading to inefficient ablation of some floxed alleles, such as Cpeb2. Moreover, Ucp1-Cre[Evdr] mice displayed nonspecific Cre expression, whereas neither line showed evidence of substantial autonomous Cre activity in BAT-resident macrophages. Consequently, ΔBAT[Evdr] mice experienced off-target neuronal ablation, resulting in hyperactive locomotion and reduced body weight. Although ΔBAT[YH] mice showed normal locomotor activity and body weight, they had a modest weight gain and altered glucose homeostasis only after high-fat-diet feeding. In conclusion, novel Ucp1-Cre knock-in mouse showed specificity and efficiency for gene manipulation in brown adipocytes, highlighting its application in generating BAT-specific knockout and BAT-depleted mouse models.NEW & NOTEWORTHY A novel Ucp1-CreYH knock-in mouse exhibits improved efficiency and specificity of Cre recombinase activity in brown adipose tissue (BAT) compared with the widely used Ucp1-CreEvdr line, which showed nonspecific Cre activity in many organs. A BAT-deprived mouse model, generated with this Cre line, showed normal locomotion and body weight, but altered body weight and glucose homeostasis after high-fat feeding, validating its use for BAT-specific gene manipulation.},
}

Animals
*Uncoupling Protein 1/genetics/metabolism
*Adipose Tissue, Brown/metabolism
Mice
*Integrases/genetics/metabolism
Mice, Transgenic
*Gene Knock-In Techniques/methods
Thermogenesis/genetics
Male
CRISPR-Cas Systems
Mice, Inbred C57BL
*Gene Targeting/methods
Female

@article {pmid41914367,
year = {2026},
author = {Lu, Q and Ye, C and Chen, R and Xing, Z and Liu, Z and Zeng, F and Gong, J and Gao, Y and Sun, X and Tang, S and Song, Y},
title = {High-Entropy Alloy Synergized with Gene Editing for Cocktail-Sensitized Radioimmunotherapy of Lung Metastases.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {25},
pages = {e22618},
doi = {10.1002/adma.202522618},
pmid = {41914367},
issn = {1521-4095},
support = {22477056//National Natural Science Foundation of China/ ; 82272138//National Natural Science Foundation of China/ ; 22375089//National Natural Science Foundation of China/ ; 82572370//National Natural Science Foundation of China/ ; 2024300315//Fundamental Research Funds for the Central Universities/ ; 2019YFA0709200//National Key R&D Program/ ; BE2021373//Jiangsu Province Key R&D Program/ ; 5431ZZXM2304//State Key Laboratory of Analytical Chemistry for Life Science/ ; },
mesh = {*Lung Neoplasms/secondary/therapy/radiotherapy/pathology ; *Gene Editing ; Animals ; Humans ; *Alloys/chemistry/pharmacology ; Cell Line, Tumor ; Mice ; CRISPR-Cas Systems ; Palladium/chemistry ; Platinum/chemistry ; Gold/chemistry ; },
abstract = {Radiotherapy (RT) eliminates cancer cells either through direct DNA damage induced by ionizing radiation or indirectly by generating cytotoxic reactive oxygen species (ROS) via radiolysis. However, high-dose radiation often triggers DNA repair mechanisms, undermining therapeutic efficacy and causing damage to surrounding healthy tissues. Thus, enhancing anti-tumor effects at lower doses while minimizing normal tissue damage and improving safety remains a key challenge in advancing RT technologies. To tackle these issues, we developed an RT-sensitizing platform, referred to as HAEPRC, which integrates a novel high-entropy alloy (HEA) composed of gold (Au), bismuth (Bi), platinum (Pt), silver (Ag), and palladium (Pd), a CRISPR/Cas9 gene-editing system, and tumor cell membranes (CM) for enhanced home-targeting and biocompatibility. We demonstrated that HAEPRC exhibits exceptional dose enhancement factors (DEFs), significantly boosting RT sensitization and improving RT-induced immunotherapeutic outcomes. Furthermore, the gene-editing system modulates the cell cycle, transforming RT-resistant cancer cells into RT-sensitive ones and further amplifying RT efficacy. Additionally, Pd-mediated bioorthogonal catalysis activates immune adjuvant production, enhancing immune responses and reinforcing anti-tumor immunity. Collectively, these features synergistically promote an enhanced abscopal immune effect, inhibiting lung cancer growth and metastasis, and providing a promising strategy to improve the efficacy and safety of RT.},
}

*Lung Neoplasms/secondary/therapy/radiotherapy/pathology
*Gene Editing
Animals
Humans
*Alloys/chemistry/pharmacology
Cell Line, Tumor
Mice
CRISPR-Cas Systems
Palladium/chemistry
Platinum/chemistry
Gold/chemistry

@article {pmid41916134,
year = {2026},
author = {Buakaew, T and Thaiwong, R and Inthanachai, T and Palaga, T and Weissman, D and Suppipat, K and Ausavarungnirun, C and Tawinwung, S},
title = {Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.},
journal = {Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie},
volume = {198},
number = {},
pages = {119300},
doi = {10.1016/j.biopha.2026.119300},
pmid = {41916134},
issn = {1950-6007},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Messenger/genetics ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cryopreservation ; Gene Knockout Techniques ; *Receptors, Antigen, T-Cell/genetics ; Antigens, CD19/genetics ; Cell Survival ; Transfection ; Gene Editing ; },
abstract = {BACKGROUND: Autologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in hematologic malignancies but remain limited by complex manufacturing processes. Allogeneic, off-the-shelf CAR T cells derived from healthy donors represent a promising alternative; however, safe implementation requires elimination of endogenous T-cell receptor (TCR) expression and flexible CAR expression strategies.

OBJECTIVE: This study aimed to develop an optimized manufacturing workflow for allogeneic CAR T cells by combining CRISPR/Cas9-mediated TCR knockout with mRNA-based CAR expression, and to evaluate cryopreservation strategies enabling on-demand CAR T-cell generation.

METHODS: Healthy donor T cells were edited at the TRAC locus using CRISPR/Cas9 to generate TCR-deficient T cells. These cells were cryopreserved and subsequently transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, cell viability, immunophenotype, cytokine secretion, and antigen-specific cytotoxicity were assessed under different cryopreservation-transfection conditions.

RESULTS: TCR knockout T cells exhibited efficient TCR disruption with reduced alloreactive proliferation. CD117 mRNA CAR T cells derived from TCR-deficient T cells demonstrated CAR expression kinetics, immunophenotypic profiles, and antigen-specific cytotoxicity comparable to wild-type CAR T cells. Evaluation of two cryopreservation strategies revealed that cryopreservation prior to mRNA electroporation preserved cell viability, phenotype, and cytotoxic function, whereas cryopreservation after mRNA transfection was associated with reduced functional activity. The optimized protocol was successfully extended to CD19- and BCMA-targeting CAR mRNAs.

CONCLUSION: Collectively, these findings establish a modular platform for producing allogeneic CAR T cells using mRNA technology, offering a practical approach for rapid, on-demand CAR T-cell therapy.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA, Messenger/genetics
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/metabolism
Cryopreservation
Gene Knockout Techniques
*Receptors, Antigen, T-Cell/genetics
Antigens, CD19/genetics
Cell Survival
Transfection
Gene Editing

@article {pmid41933706,
year = {2026},
author = {Wang, Q and Tang, J and Jiang, Y and Jiang, X and Wang, Y and Liu, W and Qin, L},
title = {Temperature-induced pupal pigmentation in Antheraea pernyi is affected by ApADC through the regulation of NBAD biosynthesis.},
journal = {Insect biochemistry and molecular biology},
volume = {191},
number = {},
pages = {104550},
doi = {10.1016/j.ibmb.2026.104550},
pmid = {41933706},
issn = {1879-0240},
mesh = {Animals ; Pupa/metabolism/growth & development/genetics/physiology ; *Pigmentation/genetics ; *Moths/genetics/metabolism/growth & development/physiology ; Temperature ; *Insect Proteins/metabolism/genetics ; *Dopamine/analogs & derivatives/biosynthesis/metabolism ; CRISPR-Cas Systems ; },
abstract = {The Chinese oak silkworm, Antheraea pernyi, exhibits temperature-dependent pupal pigmentation, forming black pupae at 23 °C and yellow pupae at 29 °C. To address this, we elucidated the molecular mechanism of this phenotypic plasticity by integrating multi-omics analyses, RNA interference, and CRISPR/Cas9. We identified N-β-alanyl dopamine (NBAD) as essential for yellow pigmentation. Transcriptomic and metabolomic analyses revealed temperature-dependent regulation of A. pernyi aspartate 1-decarboxylase (ApADC) and NBAD synthase (Apebony). This regulation correlated with elevated β-alanine/aspartic acid in yellow pupae and enriched dopamine/N-acetyldopamine (NADA) in black pupae. Beta-alanine injection at 18 °C induced a dose-dependent transition to yellow, while RNAi of ApADC or Apebony at 29 °C triggered melanization. By establishing the first CRISPR/Cas9 platform for A. pernyi, we generated ApADC mutants with disrupted GadA domains, which developed black pupae even at 29 °C; this phenotype was rescued by β-alanine supplementation. Our results show that ApADC acts as a central temperature-responsive regulator of pupal pigmentation by modulating NBAD biosynthesis. These findings provide mechanistic insights into melanization and environmental adaptation in an important lepidopteran and are expected to guide the targeted breeding of stable genetic lines for the sustainable development of the A. pernyi industry.},
}

Animals
Pupa/metabolism/growth & development/genetics/physiology
*Pigmentation/genetics
*Moths/genetics/metabolism/growth & development/physiology
Temperature
*Insect Proteins/metabolism/genetics
*Dopamine/analogs & derivatives/biosynthesis/metabolism
CRISPR-Cas Systems

@article {pmid42072072,
year = {2026},
author = {Bartusik-Aebisher, D and Justin Raj, DR and Aebisher, D},
title = {Nanomaterial-Based Therapeutic Delivery: Integrating Redox Biology, Genetic Engineering, and Imaging-Guided Treatment.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {15},
number = {4},
pages = {},
doi = {10.3390/antiox15040430},
pmid = {42072072},
issn = {2076-3921},
abstract = {Nanomaterials are emerging versatile platforms for therapeutic delivery, as they offer precise control over drug, antioxidant, and genetic payload transport across biological barriers. Inorganic, organic, hybrid, and biomimetic systems are the major classes of nanomaterials, which all have different physicochemical properties such as size, surface charge, and surface functionalization. These properties collectively influence stability, biodistribution, cellular uptake, and release kinetics. Engineering strategies are increasingly using stimuli-responsive designs that are triggered by pH, reactive oxygen species (ROS), and intracellular redox gradients to perform spatially and temporally controlled delivery. Antioxidant and redox-modulating nanocarriers are of great importance as they overcome the limited bioavailability and nonspecific activity of conventional antioxidants by improving stability, targeting oxidative microenvironments, and allowing for regulated release. Improvements in lipid, polymeric, and inorganic nanoplatforms have also developed gene delivery applications, including siRNA, mRNA, and CRISPR/Cas systems, to provide better cytosolic release and precise therapeutics. When diagnostic imaging is integrated with therapy through theranostic nanoparticles, real-time monitoring and personalized intervention are possible. Safety, scalable manufacturing, and regulatory alignment are some challenges that show the need for standardization and translational procedures to utilize the potential of theranostic nanomedicine.},
}

@article {pmid42074014,
year = {2026},
author = {Li, Y and Ma, S and Fei, T},
title = {CRISPR Applications in Alzheimer's Disease: From High-Throughput Genetic Screening to Precision Editing and CNS Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083371},
pmid = {42074014},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; tau Proteins/genetics/metabolism ; *Genetic Testing/methods ; High-Throughput Screening Assays ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Alzheimer's disease is a devastating progressive neurodegenerative disorder characterized by extracellular amyloid-beta plaques and intracellular tau tangles. Despite recent advancements in amyloid-beta-targeting immunotherapies, achieving safe and definitive disease control remains a profound clinical challenge. The CRISPR/Cas9 system has emerged as a powerful technology for precision neurogenetics, offering significant potential to address the fundamental questions behind Alzheimer's disease. This comprehensive review delineates the trajectory of CRISPR applications in Alzheimer's disease research and therapeutics. First, we explore the integration of CRISPR in engineering high-fidelity in vitro models, such as isogenic induced pluripotent stem cells and three-dimensional cerebral organoids, alongside advanced in vivo mammalian models. Second, we examine how these platforms facilitate unbiased high-throughput genetic screening to uncover molecular underpinnings regulating tau, lipid metabolism, and neuroinflammation. Third, we critically evaluate precision editing strategies targeting core risk genes (APP, MAPT, APOE, and TREM2), explicitly highlighting the severe physiopathological trade-offs between therapeutic efficacy and loss-of-function toxicity. Finally, we address the ultimate translational bottlenecks impeding clinical application. By dissecting the packaging limits of adeno-associated viral vectors and the physical barricade of the blood-brain barrier, we underscore the necessity of transitioning toward next-generation base editors and non-viral lipid nanoparticles to realize safe and efficacious in vivo clinical gene therapies against Alzheimer's disease.},
}

*Alzheimer Disease/genetics/therapy
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
Genetic Therapy/methods
tau Proteins/genetics/metabolism
*Genetic Testing/methods
High-Throughput Screening Assays
Induced Pluripotent Stem Cells/metabolism

@article {pmid42074029,
year = {2026},
author = {O'Hanlon Cohrt, K and O'Dea, S},
title = {Clinical Trial Landscape of Gene-Edited Autologous Hematopoietic Stem Cells for Hemoglobinopathies and Immunodeficiencies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083384},
pmid = {42074029},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Hematopoietic Stem Cell Transplantation/methods ; *Hematopoietic Stem Cells/metabolism ; Transplantation, Autologous ; Clinical Trials as Topic ; *Immunologic Deficiency Syndromes/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Allogeneic hematopoietic cell transplantation (HCT) has been used for decades to treat certain malignant and non-malignant hematological conditions, but challenges remain. Increased understanding of disease mechanisms and recent developments in genome editing have enabled alternative strategies utilizing gene-edited autologous HCT and many of these have progressed to the clinic. We present here a comprehensive review of clinical trials of gene-edited autologous hematopoietic stem cells for the treatment of hemoglobinopathies and immunodeficiencies. Searches of major international clinical trial registries were carried out using specific key words. In total, 44 interventional clinical trials investigating gene-edited autologous stem cell therapies were identified, with CASGEVY (exagamglogene autotemcel) being the only product approved to date. Hemoglobinopathies were the most common indication (n = 37) followed by immunodeficiencies (n = 4), with single trials in HIV-1 infection, pyruvate kinase deficiency and limb-girdle muscular dystrophy. Gene-editing strategies fall into three categories: disruption of the BCL11A erythroid enhancer, editing of the γ-globin promoter and direct correction or disruption of disease-relevant genes. CD34[+] hematopoietic stem and progenitor cells are the most common cell types edited, and CRISPR-Cas9 is the most widely used gene-editing modality. While results are encouraging, efficient intracellular delivery of gene-editing tools, editing efficiencies and off-target editing remain challenges for the field.},
}

Humans
*Gene Editing/methods
*Hemoglobinopathies/therapy/genetics
*Hematopoietic Stem Cell Transplantation/methods
*Hematopoietic Stem Cells/metabolism
Transplantation, Autologous
Clinical Trials as Topic
*Immunologic Deficiency Syndromes/therapy/genetics
CRISPR-Cas Systems
Genetic Therapy/methods

@article {pmid42074108,
year = {2026},
author = {An, L and Xu, Z and Zhang, X},
title = {Self-Assembling Short Peptide Carriers for Gene Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083464},
pmid = {42074108},
issn = {1422-0067},
support = {12574240//National Natural Science Foundation of China/ ; 12174462//National Natural Science Foundation of China/ ; IS24073//Beijing Natural Science Foundation/ ; 2025XYCM83//Organized Research Program in Minzu University of China/ ; },
mesh = {Humans ; *Gene Transfer Techniques ; *Peptides/chemistry ; Animals ; Genetic Therapy/methods ; Gene Editing ; *Drug Carriers/chemistry ; CRISPR-Cas Systems ; },
abstract = {Gene therapy relies on safe and efficient delivery systems, yet traditional viral vectors and synthetic polymers often fail to meet these requirements due to immunogenicity and biocompatibility concerns. This review highlights self-assembling short peptides as a highly programmable and biocompatible non-viral platform uniquely positioned to overcome these translational bottlenecks. To provide a comprehensive overview of next-generation gene delivery, we systematically trace the trajectory from fundamental chemistry to clinical applications. First, we elucidate the supramolecular interactions and mechanisms driving peptide-nucleic acid co-assembly. Second, we outline concrete design strategies, detailing how sequence engineering and environmental responsiveness dictate the formation of optimized nanomorphologies. Third, we critically analyze how these nanocarriers navigate critical physiological and intracellular barriers, with a specific focus on cellular uptake, endosomal escape, and cargo release. Finally, we demonstrate the platform's versatility in emerging frontiers, particularly mRNA vaccines and CRISPR/Cas9 gene editing. We conclude by identifying current obstacles to clinical translation and proposing future directions centered on multifunctional integration and stimuli-responsive design.},
}

Humans
*Gene Transfer Techniques
*Peptides/chemistry
Animals
Genetic Therapy/methods
Gene Editing
*Drug Carriers/chemistry
CRISPR-Cas Systems

@article {pmid42074129,
year = {2026},
author = {Lan, Z and Tian, M and Liu, J and Shi, W and Chen, T and Ma, Q and Jin, B and Zhao, Y and Zhang, H and Lai, CJ and Cui, G},
title = {Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083485},
pmid = {42074129},
issn = {1422-0067},
support = {CI2021A05051//China Academy of Chinese Medical Sciences/ ; },
mesh = {*Salvia miltiorrhiza/genetics/metabolism ; *CRISPR-Cas Systems ; *Abietanes/biosynthesis ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Phylogeny ; *Hydroxymethylglutaryl CoA Reductases/genetics/metabolism ; },
abstract = {3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) serves as a key rate-limiting enzyme in the mevalonate pathway and plays a central regulatory role in the biosynthesis of tanshinones. To date, four HMGR family members (SmHMGR1-4) have been identified in Salvia miltiorrhiza. Here, we cloned and identified a novel member, SmHMGR5, by integrating multiple genomic datasets. Genomically, SmHMGR5 formed an inverted repeat with SmHMGR3 (98.04% homology) and phylogenetically clustered with SmHMGR2. Based on the expression patterns of the five HMGR genes, we further generated SmHMGR2 and SmHMGR5 knockout mutants using CRISPR/Cas9 technology and compared their effects on the accumulation of 12 tanshinones and 4 phenolic acids via UPLC-MS-based metabolomic analysis. Knockout of SmHMGR2 significantly suppressed the accumulation of seven tanshinones, whereas SmHMGR5 knockout downregulated only three tanshinones, and neither mutation affected phenolic acids. Notably, the major compound tanshinone IIA remained stable across different mutants, but tanshinone IIB was markedly reduced upon SmHMGR2 knockout, suggesting complex regulatory mechanisms in tanshinone biosynthesis. These findings provide new insights into the biosynthetic network of tanshinones and establish a theoretical foundation for metabolic engineering strategies aimed at enhancing the production of bioactive constituents in S. miltiorrhiza.},
}

*Salvia miltiorrhiza/genetics/metabolism
*CRISPR-Cas Systems
*Abietanes/biosynthesis
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Phylogeny
*Hydroxymethylglutaryl CoA Reductases/genetics/metabolism

@article {pmid42074203,
year = {2026},
author = {Guo, B},
title = {CRISPR Interference to Inhibit Oncogenes for Cancer Therapy.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083564},
pmid = {42074203},
issn = {1422-0067},
support = {1R01CA293945-02/NH/NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Oncogenes/genetics ; Animals ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {CRISPR interference (CRISPRi), a programmable transcriptional repression technology derived from nuclease-deficient CRISPR-Cas systems, has emerged as a powerful method for selectively inhibiting oncogene expression without altering the genomic DNA. This feature offers a major advantage over other oncogene targeting technologies such as CRISPR-mediated gene knockout, mRNA inhibition by siRNA or miRNA, or small-molecule inhibitors of the proteins encoded by the oncogenes, especially in cancers driven by transcriptional dysregulation or otherwise undruggable oncogenes. Here, I present a comprehensive review of CRISPRi mechanisms, delivery strategies, and preclinical applications in oncology (including advances in targeting core oncogenic drivers like MYC and KRAS). The advantages of CRISPRi as well as in vivo validation of CRISPRi-mediated tumor suppression are discussed. Finally, I outline translational challenges and future directions for incorporating CRISPRi into precision cancer therapies. The accumulated evidence suggests that CRISPRi could become a cornerstone for next-generation gene-regulatory therapeutics.},
}

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

@article {pmid42074279,
year = {2026},
author = {Sterckel, S and Chávez Martínez, IL and Schwach, V},
title = {CRISPR and the Future of Cardiac Disease Therapy: A New Genetic Frontier.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083641},
pmid = {42074279},
issn = {1422-0067},
support = {10250042110011//ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Heart Diseases/therapy/genetics ; *Genetic Therapy/methods ; Animals ; },
abstract = {CRISPR technologies are transforming cardiovascular therapy development by creating an increasingly seamless pipeline from potential target discovery to clinical translation. What began as a genome-editing tool has evolved into a versatile platform that enables researchers to precisely interrogate and modulate cardiac biology with tools such as base- and prime-editors, and CRISPR inhibition and activation. In this review, we follow the use of CRISPR across the stages of biomedical research through to bench-to-bedside application. This review begins by addressing how genome-wide and focused CRISPR screens discover developmental regulators, disease drivers, and drug-response pathways, making the first steps in identifying therapeutic targets. We then explore how CRISPR engineering creates progressively more relevant disease model systems to validate mechanisms of disease and test interventions, helping bridge the translational gaps between the lab and the clinic. Finally, we consider how CRISPR technologies are beginning to enter cardiovascular clinical trials, while highlighting the key challenges that still limit this translation. By linking the latest advances of modern CRISPR platforms to the stages of therapeutic development, this review highlights how CRISPR technology is reshaping the pipeline from molecular insight to clinical innovation in cardiac disease.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Heart Diseases/therapy/genetics
*Genetic Therapy/methods
Animals

@article {pmid42074580,
year = {2026},
author = {Gonzalez-Chavez, Z and Siddiqui, MA and Ganesan, S and Desai, SA},
title = {A CRISPR-Based Mutagenesis Strategy for Examining CLAG3 Helix 44 Contribution to Malaria Parasite Nutrient Uptake Channels.},
journal = {Genes},
volume = {17},
number = {4},
pages = {},
doi = {10.3390/genes17040462},
pmid = {42074580},
issn = {2073-4425},
support = {ZIA AI000882-25//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Protozoan Proteins/genetics/metabolism/chemistry ; *Plasmodium falciparum/genetics/metabolism ; Mutagenesis ; CRISPR-Cas Systems ; Erythrocytes/parasitology/metabolism ; Humans ; *Ion Channels/genetics/metabolism ; Nutrients/metabolism ; },
abstract = {Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed α-helix 44 (α-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without α-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of α-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized α-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a α-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target.},
}

*Protozoan Proteins/genetics/metabolism/chemistry
*Plasmodium falciparum/genetics/metabolism
Mutagenesis
CRISPR-Cas Systems
Erythrocytes/parasitology/metabolism
Humans
*Ion Channels/genetics/metabolism
Nutrients/metabolism

@article {pmid42075136,
year = {2026},
author = {Zhang, E and Yan, J and Du, J and Chu, X and Chen, D},
title = {Application and Research Prospects of CRISPR/Cas Gene Editing Technology in Lactic Acid Bacteria.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/microorganisms14040739},
pmid = {42075136},
issn = {2076-2607},
abstract = {Lactic acid bacteria (LAB) are pivotal microorganisms in the food industry. Current approaches for functional gene validation and trait improvement in LAB primarily rely on traditional gene editing and homologous recombination techniques. These methods are often cumbersome, inefficient, and time-consuming, hindering the rapid and precise customization of strains. This limitation has, to some extent, constrained the rapid selection and industrial application of functional LAB strains. The engineering of LAB through gene editing technologies has significantly advanced both fundamental and applied research. Among these, CRISPR/Cas gene editing has successfully achieved precise modification of multiple genes in various LAB species. Compared to conventional methods, it offers superior editing efficiency and lower operational costs, opening new avenues for functional gene identification and genetic improvement in LAB. However, the application of exogenous CRISPR/Cas systems in LAB faces technical challenges such as high off-target rates, chromosomal abnormalities, and cytotoxicity. The development of endogenous CRISPR/Cas-based editing tools for LAB provides novel pathways for precise regulation, rational design, and flexible application. This paper first outlines the structural components and mechanistic principles of CRISPR/Cas gene editing tools. It then explores the research progress and applications of both endogenous and exogenous CRISPR/Cas systems in LAB. Finally, it provides an outlook on the future application of CRISPR/Cas gene editing technology in LAB, offering a reference for its implementation in this field. The advent of gene editing technologies has significantly propelled functional gene validation and trait improvement in lactic acid bacteria (LAB), thereby advancing both fundamental research and industrial applications. Notably, the CRISPR/Cas system has emerged as a transformative tool enabling precise genetic modification in diverse LAB species, offering marked improvements in editing efficiency and cost reduction relative to conventional approaches. CRISPR/Cas-based editing strategies in LAB are broadly classified into exogenous and endogenous systems. Exogenous systems operate independently of the host's native immune repertoire, conferring the advantages of broad strain applicability and high editing efficiency. These systems have been successfully deployed for functional gene characterization, metabolic pathway engineering, such as augmenting antimicrobial production, and probiotic safety enhancement via virulence gene deletion. Conversely, endogenous systems leverage the intrinsic CRISPR/Cas machinery of LAB, offering superior biocompatibility and minimized off-target risks. Notable applications include precise gene knockout and integration using the native Type I-E system in Lacticaseibacillus paracasei. This review provides a concise overview of CRISPR/Cas system architecture and mechanisms, followed by a systematic synthesis of research progress and applications for both exogenous and endogenous systems in LAB. Finally, future directions are outlined to guide the continued development and application of CRISPR/Cas technologies in this field.},
}

@article {pmid42075335,
year = {2026},
author = {Liu, Q and Qiu, Z and Yao, M and Jiao, B and Zhou, Y and Li, C and Liu, H and Xin, L},
title = {Progress of Rapid Detection Technology for Aquatic Microorganisms: A Comprehensive Review.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/microorganisms14040939},
pmid = {42075335},
issn = {2076-2607},
support = {No. 2023YFD2403000//National Key Research and Development Program of China/ ; 2024KJN029//the Ministry of Science and Technology of the People's Republic of China, Shandong Provincial University Youth Innovation Team Project/ ; },
abstract = {Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020-2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption-from days to less than one hour-while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements.},
}

@article {pmid42075363,
year = {2026},
author = {Di Pinto, A and Forte, V and D'Attilia, C and Possenti, M and Felici, B and Augelletti, F and Sessa, G and Carabelli, M and Morelli, G and Frugis, G and D'Orso, F},
title = {A Rapid Hairy Root-Based Platform for CRISPR/Cas Optimization and Guide RNA Validation in Lettuce.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/plants15081161},
pmid = {42075363},
issn = {2223-7747},
support = {CN00000022//European Union Next-GenerationEU/ ; A0375E0166//Lazio Innova/ ; },
abstract = {Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, 'Saladin' and 'Osiride', identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing.},
}

@article {pmid42079440,
year = {2026},
author = {Yang, L and Luo, R and Zhou, W and Yin, P and Feng, Y and Zhang, Y},
title = {Recent advances in noncanonical inhibition mechanisms of anti-CRISPR proteins.},
journal = {mLife},
volume = {5},
number = {2},
pages = {133-147},
pmid = {42079440},
issn = {2770-100X},
abstract = {The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage-host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.},
}

@article {pmid42079647,
year = {2026},
author = {Requejo Cier, CJ and Valentini, N and Boudreau, G and Delisle, JS and Lamarche, C},
title = {Engineering human Tregs to resist tacrolimus via FKBP12 gene editing.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1756624},
pmid = {42079647},
issn = {1664-3224},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/drug effects/metabolism ; *Tacrolimus/pharmacology ; *Tacrolimus Binding Protein 1A/genetics/metabolism ; *Gene Editing ; Cell Proliferation/drug effects ; CRISPR-Cas Systems ; *Immunosuppressive Agents/pharmacology ; Interleukin-2 ; Lymphocyte Activation/drug effects ; Cells, Cultured ; },
abstract = {Regulatory T cells (Tregs) are essential for immune tolerance and are under active development as cell therapy in transplantation. However, the widespread use of the calcineurin inhibitor tacrolimus may inadvertently suppress Treg proliferation and activation, undermining their therapeutic potential. Tacrolimus binds to the FKBP12 protein in T cells, forming a complex that blocks calcineurin-NFAT signaling and suppresses IL-2 gene transcription, thereby inhibiting T cell activation. In this study, we investigated whether deleting FKBP12 in human Tregs could prevent tacrolimus-mediated suppression. Using CRISPR-Cas9 gene editing, FKBP12 was knocked out in ex vivo expanded human Tregs, which were then cultured for seven days with tacrolimus (10 ng/mL) or control, under varying IL-2 concentrations (100-500 IU/mL). We observed that tacrolimus significantly reduced the proliferation of control Tregs, even in conditions with 500 IU/mL IL-2, whereas FKBP12-knockout Tregs maintained robust proliferation comparable to untreated cells. We found no discernible changes in Treg phenotype or stability following FKBP12 deletion or tacrolimus exposure: edited Tregs retained normal expression of the lineage-defining marker FOXP3, displayed a global transcriptomic profile nearly indistinguishable from controls, and were similarly suppressive, indicating that they remained bona fide Tregs. These findings demonstrate that the antiproliferative effect of tacrolimus on Tregs is critically dependent on FKBP12, mirroring its mechanism in conventional T cells. By genetically uncoupling tacrolimus from its target in Tregs, this approach suggests a strategy to preserve Treg numbers during tacrolimus-based immunosuppression in transplant recipients, potentially enhancing Treg-based therapies for transplantation tolerance.},
}

Humans
*T-Lymphocytes, Regulatory/immunology/drug effects/metabolism
*Tacrolimus/pharmacology
*Tacrolimus Binding Protein 1A/genetics/metabolism
*Gene Editing
Cell Proliferation/drug effects
CRISPR-Cas Systems
*Immunosuppressive Agents/pharmacology
Interleukin-2
Lymphocyte Activation/drug effects
Cells, Cultured

@article {pmid42080108,
year = {2026},
author = {Yousefian, M and Baharmast, M},
title = {Artificial Intelligence-Assisted CRISPR Gene Editing: Current Advances, Clinical Challenges, and Future Directions in Precision Medicine.},
journal = {Avicenna journal of medical biotechnology},
volume = {18},
number = {1},
pages = {3-15},
pmid = {42080108},
issn = {2008-2835},
abstract = {Recent advances in Artificial Intelligence (AI) have profoundly transformed the field of genome editing, particularly through integration with the Clustered Regularly Inter-spaced Short Palindromic Repeats (CRISPR) technology. This review highlights how AI-driven computational models are reshaping guide RNA (gRNA) design, off-target prediction, and editing precision in CRISPR-Cas systems. A PRISMA-informed literature survey was conducted using PubMed, Scopus, EMBASE, and Google Scholar databases to identify studies exploring AI-assisted CRISPR applications in gene therapy and biomedical research. The results demonstrate that deep learning, machine learning, and reinforcement learning approaches significantly enhance prediction accuracy, algorithmic efficiency, and translational potential across genetic diseases such as β-thalassemia, muscular dystrophy, and cancer. Moreover, ethical challenges, algorithmic bias, and data security concerns remain critical barriers to clinical adoption. This review also discusses the emerging landscape of AI-assisted CRISPR research in Iran, emphasizing national progress, infrastructural constraints, and future opportunities. Overall, the convergence of AI and CRISPR technologies promises to advance precision medicine by accelerating the development of personalized, efficient, and ethically responsible genome-editing solutions.},
}

@article {pmid42080254,
year = {2026},
author = {Cho, SW and Kim, T and Yang, J and Byun, G and Seo, SW},
title = {Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag422},
pmid = {42080254},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2024-0035256//Ministry of Science and ICT/ ; RS-2025-02214910//Ministry of Science and ICT/ ; RS-2025-02309093//Ministry of Science and ICT/ ; RS-2024-00345885//Korean government/ ; SRFC-MA1901-11//Samsung Research Funding & Incubation Center of Samsung Electronics/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli Proteins/genetics ; },
abstract = {The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10-8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic Escherichia coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.},
}

*Gene Editing/methods
*Escherichia coli/genetics
*CRISPR-Cas Systems
Genes, Essential
*Clustered Regularly Interspaced Short Palindromic Repeats
Escherichia coli Proteins/genetics

@article {pmid42080258,
year = {2026},
author = {Yang, H and Shen, B and Wang, Y and Liu, J and Zhou, F and Liu, M and Li, J and Fan, J and Ding, S and Guo, J and Zhang, J and Li, X},
title = {Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag414},
pmid = {42080258},
issn = {1362-4962},
support = {82202649//National Natural Science Foundation of China/ ; 82202649//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0007//Chongqing Natural Science Foundation Joint Fund for Innovation and Development/ ; CSTB2023NSCQ-MSX0897//Natural Science Foundation of Chongqing/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Single-Stranded/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics ; HIV-1/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise regulation of the trans-cleavage activity of CRISPR/Cas12a has substantially expanded its utility in molecular diagnostics. However, existing strategies rely predominantly on systems with freely diffusing components, necessitating intricate CRISPR RNA (crRNA) designs or specialized chemical modifications, which hinder their simplicity and broader applicability. Here, we demonstrate that the activity of spatially confined Cas12a on fluid membranes (CAS-FLIER) can be facilely modulated by simply adjusting the length of crRNA and the duplex-strand reporters. We reveal that fine-tuning the movement range of membrane-Cas12a and the accessibility of the reporter to Cas12a enables precise, scalable control over trans-cleavage activity. As a proof of concept, we show that the activity of confined Cas12a can be co-activated by single-stranded DNA (ssDNA) and RNA inputs, a capability that remains unattainable in conventional freely diffusing systems. Furthermore, by incorporating a DNA reverse-transcriptor into the CAS-FLIER system, we achieve one-pot, highly sensitive detection of HIV RNA, supporting accurate diagnosis of HIV infection. Notably, this assay is compatible with a lateral-flow format for direct visual readout, highlighting its potential as a point-of-care diagnostic tool for HIV. Collectively, our findings shed new light on modulating Cas12a activity, advancing its applications in molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
DNA, Single-Stranded/genetics/metabolism
*Bacterial Proteins/metabolism/genetics
HIV-1/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42080260,
year = {2026},
author = {Luo, W and Wu, Y and Ni, D and Zhang, L and Zhang, Y and Han, X and Zhang, Y and Pu, J and He, Y and Yin, N and Wang, W and Huang, R and Guo, Y and Sun, Y and Xie, G},
title = {A universal and orthogonal safety valve for CRISPR/Cas12a without chemical modification or external stimulation.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag436},
pmid = {42080260},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; 82501041//National Natural Science Foundation of China/ ; BJRC202410//Chongqing Medical University/ ; GZC20251421//National Postdoctoral Researcher Support Program/ ; CSTB2025NSCQ-GPX1184//Chongqing Natural Science Foundation General Project/ ; HBRC202404//Chongqing National Reserve Talent Program in Health and Wellness/ ; CSTB2025NSCQ-JQX0016//Chongqing Outstanding Youth Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/antagonists & inhibitors/chemistry ; Humans ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; RNA/genetics/antagonists & inhibitors/chemistry ; Proprotein Convertase 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {CRISPR/Cas-based gene editing technologies have achieved remarkable progress over the past decade, yet their broad practical applications remain limited by safety concerns. Although regulatory strategies applied before or during CRISPR/Cas activation have substantially improved sequence, temporal, and spatial specificity, persistent activity of already activated Cas nucleases may still increase the risk of uncontrolled editing. Therefore, an effective post-activation control strategy is urgently needed. Here, we report a modification- and stimulation-free RNA inhibitor (iRNA) that functions as a post-activation safety valve for CRISPR/Cas12a. By exploiting Cas12a's allosteric sensitivity and the thermodynamic and kinetic programmability of nucleic acid strand displacement, iRNA drives already activated Cas12a ribonucleoproteins back to an inactive state, enabling universal, sequence-programmable, and orthogonal post-activation inhibition within the validated Cas12a framework. Experiments and simulations elucidate the mechanistic basis of iRNA-mediated strand displacement and demonstrate its high inhibitory efficiency, reversible cyclic control, compatibility, expandability, orthogonality, and universality. Importantly, iRNA also acts as a programmable, autonomously operating safety valve in cells, suppressing uncontrolled editing while preserving PCSK9 gene knockout. With its simple design, excellent biocompatibility, and autonomous intracellular expression, iRNA provides a foundation for next-generation controllable CRISPR systems and holds broad potential for precision therapeutics, cell therapy, and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*CRISPR-Associated Proteins/metabolism/genetics/antagonists & inhibitors/chemistry
Humans
*Endodeoxyribonucleases/genetics/metabolism/chemistry
*Bacterial Proteins/genetics/metabolism
RNA/genetics/antagonists & inhibitors/chemistry
Proprotein Convertase 9/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid42080263,
year = {2026},
author = {Feng, W and Hu, J and Zhang, H and Le, XC},
title = {A kinetic approach for mapping seed regions of CRISPR ribonucleoprotein and improving specificity.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag413},
pmid = {42080263},
issn = {1362-4962},
support = {TIPS CBRF2-2023-001//Canadian Biomedical Research Fund/ ; RGPIN-2024-05974//Natural Sciences and Engineering Research Council of Canada/ ; 22506171//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *Ribonucleoproteins/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Binding ; Binding Sites ; },
abstract = {The binding of an activator (target nucleic acid) to a crRNA-Cas ribonucleoprotein (RNP) in CRISPR systems is critical to the activation, kinetics, and specificity of the CRISPR technology. Key to this activation process is the interaction between the protospacer region of the activator and the spacer region of the crRNA in the RNP complex. However, how the nucleotides in the spacer region of the crRNA contribute to the kinetics of RNP binding is not well characterized. We report here profiling of the kinetically critical regions in the process of RNP binding to activators (RNA targets). We introduced the concept and strategy of kinetic manipulators, which enabled mapping of the seed regions (6-9 nucleotides within the spacer that is sensitive to mismatches) of the CRISPR-Cas13a system, including the LbuCas13a and LwaCas13a homologs. The characterization of the binding kinetics and the introduction of kinetic manipulators provided the foundation for a new kinetic approach to improve the specificity of CRISPR techniques without sacrificing the activity. Profiling the kinetically critical regions in the CRISPR system and designing corresponding manipulators maximized the kinetic differences, between the on-target and off-target, and increased discrimination of single-nucleotide mismatches.},
}

*CRISPR-Cas Systems/genetics
Kinetics
*Ribonucleoproteins/metabolism/genetics/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Protein Binding
Binding Sites

@article {pmid42080266,
year = {2026},
author = {Purcell, J and Liu, L and Calvert, RW and Hayes, BK and Huang, C and Davidovich, C and Knott, GJ and Rosenbluh, J},
title = {DUSP11 is an RNA triphosphatase that limits PspCas13b activity by destabilizing gRNA abundance in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag412},
pmid = {42080266},
issn = {1362-4962},
support = {APP2011329//National Health and Medical Research Council/ ; //Australian Government Research Training Program Scholarship/ ; APP1175568//National Health and Medical Research Council/ ; SMRF2021-276//Snow Medical Research Foundation/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Dual-Specificity Phosphatases/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; HEK293 Cells ; *Mitogen-Activated Protein Kinase Phosphatases/genetics/metabolism ; RNA Stability ; Animals ; Gene Knockout Techniques ; RNA Polymerase III/metabolism/genetics ; },
abstract = {The CRISPR-Cas13 system enables programmable RNA targeting with potential applications in therapeutics and research. However, while PspCas13b mediates efficient RNA knockdown following transient transfection, stable lentiviral delivery results in minimal activity, limiting its utility. Here, we performed a genome-wide CRISPR-Cas9 knockout screen to identify mammalian factors that restrict PspCas13b activity. We discovered that DUSP11, an RNA triphosphatase, suppresses PspCas13b function by dephosphorylating the 5'-triphosphate of Pol III-transcribed guide RNAs (gRNAs), triggering their degradation. DUSP11 knockout increased gRNA levels 2.5-4-fold and enhanced PspCas13b-mediated knockdown across multiple cell lines. This enhancement was sustained for at least 27 days and enabled targeting of endogenous transcripts previously refractory to PspCas13b. Our findings reveal an unexpected host restriction of bacterial CRISPR systems and demonstrate that gRNA levels are a limiting factor. We provide a simple strategy to improve PspCas13b activity in mammalian cells. These results have implications for developing PspCas13b-based therapeutics and suggest that systematic identification of host factors regulating CRISPR components could enhance genome editing technologies.},
}

Humans
CRISPR-Cas Systems
*Dual-Specificity Phosphatases/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
HEK293 Cells
*Mitogen-Activated Protein Kinase Phosphatases/genetics/metabolism
RNA Stability
Animals
Gene Knockout Techniques
RNA Polymerase III/metabolism/genetics

@article {pmid42080267,
year = {2026},
author = {Pan, L and Sang, R and Xue, R and Ma, Y and Goldys, E and Deng, F},
title = {AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag430},
pmid = {42080267},
issn = {1362-4962},
support = {//Henan Province High-level Talent International Exchange Project/ ; 252300423880//Henan Provincial Natural Science Foundation/ ; //UNSW SHARP/ ; 2024/ECF1573//Cancer Institute NSW/ ; DP240103024//ARC/ ; 2030464//NHMRC/ ; 2024/ECF1573//Cancer Institute NSW/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *Ribonucleoproteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Gene Editing ; DNA, Single-Stranded/metabolism ; Models, Molecular ; Protein Multimerization ; },
abstract = {Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5'-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5'-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA-one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.},
}

*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism
*Ribonucleoproteins/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
*CRISPR-Cas Systems
Gene Editing
DNA, Single-Stranded/metabolism
Models, Molecular
Protein Multimerization

@article {pmid42080294,
year = {2026},
author = {Liu, Q and Guan, J and He, X and Xie, P and Zhao, Z and Liu, X and Lee, TY and Chiang, YC and Yao, L},
title = {EnAcrPred: A robust ensemble machine learning framework for identifying anti-CRISPR proteins.},
journal = {Protein science : a publication of the Protein Society},
volume = {35},
number = {6},
pages = {e70559},
doi = {10.1002/pro.70559},
pmid = {42080294},
issn = {1469-896X},
support = {JCYJ20230807114206014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2025A1515011753//Guangdong Province Basic and Applied Basic Research Fund/ ; 2025XAKJ0102017//Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory/ ; 20720250172//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; Gene Editing ; *Software ; },
abstract = {The identification of anti-CRISPR proteins (Acrs) is crucial for understanding the regulation of CRISPR-Cas systems and their application in gene editing. However, current experimental methods face challenges, particularly in detecting Acrs with low similarity to known protein sequences. To address these challenges, we propose EnAcrPred, an advanced prediction framework based on ensemble learning. The model combines features such as sequence composition, order correlation, and inferred structure and utilizes a stacking ensemble architecture to integrate multiple base models, which enhances both the accuracy and generalization ability of the predictions. Experimental results demonstrate that EnAcrPred achieves superior performance over existing methods across multiple evaluation metrics, further confirming its robustness. Additionally, SHapley Additive exPlanations (SHAP) value analysis identifies the key features influencing Acrs recognition. To facilitate broad adoption in practice, we developed an online platform where users can quickly obtain Acrs predictions by entering a protein sequence. EnAcrPred offers an effective solution for Acrs identification, contributing to the advancement of gene editing research and safety. The platform is accessible via the link at https://ycclab.cuhk.edu.cn/EnAcrPred/.},
}

*Machine Learning
*CRISPR-Cas Systems
Gene Editing
*Software

@article {pmid42080605,
year = {2026},
author = {Merwaiss, F and Aragonés, V and García, A and Daròs, JA},
title = {A Modified Cas9 Scaffold Allows Extension of the Virus-Induced Gene Editing Technology to the Large Potyvirus Genus.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70675},
pmid = {42080605},
issn = {1467-7652},
support = {PID2023-146418OB-I00//Spanish Ministerio de Ciencia, Innovación y Universidades/ ; FPU20/05477//Spanish Ministerio de Ciencia, Innovación y Universidades/ ; CIPROM/2022/21//Generalitat Valenciana/ ; },
abstract = {Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprise the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompasses most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (Potyvirus nicotianainsculpentis)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed us to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings may be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.},
}

@article {pmid42081667,
year = {2026},
author = {Xie, H and Geng, L and Hu, Z and Chen, F and Tan, M and Wang, D and Huang, Z and Ye, W and Chen, P and Zhu, J},
title = {Genome editing generates high oleic soybean and eliminates beany flavors.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70282},
pmid = {42081667},
issn = {1744-7909},
abstract = {Soybeans serve as excellent sources of vegetable oil, protein, and other valuable nutrients for human consumption, materials for diverse industries, including the cosmetics and medical industries, and feedstocks for animals. Nevertheless, some people do not favor soy oil or other various food products derived from soybeans, due to inadequate levels of oleic acid in the oil and the presence of undesirable grassy and beany flavors associated with oxidation products of polyunsaturated fatty acids in the seeds. In this study, we developed soybean cultivars with very high levels of oleic acid in the seeds, and without grassy and beany flavors. We achieved this by using CRISPR-Cas-SF01 to edit genes in the elite cultivar Xudou 18 (XD18), targeting two microsomal Δ-12 fatty acid desaturase 2 (GmFAD2-1A and GmFAD2-1B) and three lipoxygenase (GmLOX1, GmLOX2, and GmLOX3) genes. Our findings showed that fad2-1a/b and fad2-1a/b/lox1/2/3 plants performed similarly to XD18 plants in the field, indicating no obvious growth penalties. Overall, this research has demonstrated that the development of soybean germplasms with high levels of oleic acid and without undesirable beany flavors through gene-editing of multiple relevant genes is effective, and this endeavor can contribute to the health of a broader global consumer population.},
}

@article {pmid42082082,
year = {2026},
author = {Saliani, N and Hejazi, MS and Vahed, SZ and Aghdam, EM and Mori, Z and Hasheminejad, N and Parvar, MD and Montazersaheb, S and Ebrahimi, V},
title = {Synthetic Biology-Driven Innovations in Triple-Negative Breast Cancer: Integrating Engineering Design with Targeted Therapeutics.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2026.04.017},
pmid = {42082082},
issn = {1873-4863},
abstract = {Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited therapeutic options and poor clinical outcomes due to the absence of hormone-responsive receptors. The advent of synthetic biology, which integrates molecular biology with engineering design principles, has introduced new opportunities to develop precise and programmable therapeutic and diagnostic strategies for TNBC. Engineered immune cells, such as chimeric antigen receptor (CAR)-T constructs, can selectively recognize tumor-associated antigens and overcome immunosuppressive barriers. Synthetic gene circuits and engineered bacteria enable tumor-specific delivery of cytotoxic or immunomodulatory agents, while induced pluripotent stem cells (iPSCs) provide patient-specific platforms for disease modeling and drug screening. In parallel, CRISPR/Cas-based genome editing facilitates targeted modulation of oncogenic and tumor-suppressor networks, offering both mechanistic insights and therapeutic innovation. This review highlights current advances in synthetic biology-driven approaches for TNBC, encompassing cell-based, microbial, and nucleic acid-engineered systems. It also discusses their synergistic potential to mitigate tumor heterogeneity, enhance therapeutic specificity, and overcome drug resistance. Collectively, the intersection of synthetic biology, immuno-oncology, and precision medicine holds significant promise for next-generation, adaptive, and patient-tailored treatments for TNBC.},
}

@article {pmid41634255,
year = {2026},
author = {Liu, G and Tian, X and Ye, L and Han, P and Wang, Q and Cao, Y and Wu, Y and Lu, Y},
title = {CRISPR-Cas9-mediated uATG introduction in the 5'UTR of the Uox gene for hyperuricemia mouse models: implications for gout and metabolic disorders.},
journal = {Science China. Life sciences},
volume = {69},
number = {5},
pages = {1620-1633},
pmid = {41634255},
issn = {1869-1889},
mesh = {Animals ; *Hyperuricemia/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice ; Humans ; *5' Untranslated Regions/genetics ; Gene Editing/methods ; HEK293 Cells ; *Gout/genetics/metabolism ; *Metabolic Diseases/genetics ; Gene Knockdown Techniques ; Uric Acid/blood ; Male ; Mice, Inbred C57BL ; },
abstract = {Sequence-specific gene knockdown technologies are crucial for fundamental research and therapeutic applications. RNA interference and CRISPR interference, while extensively utilized for gene expression manipulation, face limitations due to their ectopic or transient expression. In this study, we developed a generalizable and efficient method to downregulate gene expression in human 293T cells by introducing de novo upstream ATGs (uATGs) of genes using CRISPR-Cas9-mediated genome editing. Through CRISPR library screening, in-depth sequencing, and flow cytometry analysis, we validated that the introduction of uATGs served as an effective method to suppress protein expression. Our findings further revealed that this strategy can be tailored to diminish endogenous gene expression in tumor cells without affecting the mRNA transcription levels. Importantly, by introducing a uATG into the 5' untranslated region (UTR) of the Uox gene, we successfully established a Uox-knockdown (KD) mouse model of hyperuricemia associated with metabolic disorders. This model demonstrated hyperuricemia, with serum uric acid levels that exceeded 400 µmol L[-1], along with renal dysfunction, as indicated by elevated serum creatinine and blood urea nitrogen levels. Examination of the kidneys from 8-week-old Uox-KD mice revealed abnormal histopathological characteristics, including partial dilation of Bowman's capsules and renal tubules, focal nephron collapse and necrosis, and lymphocytic infiltration. In addition, the mice exhibited lipid and glucose metabolism disorders, all while maintaining a normal lifespan. This spontaneous hyperuricemia model has potential as a valuable tool for long-term studies on hyperuricemia and gout. Taken together, we present an efficient approach for the constant suppression of specific gene expression in mammalian cells and the development of a Uox-KD mouse model of hyperuricemia via CRISPR-Cas9-mediated uATG introduction. This offers broad implications for fundamental research and therapeutic applications.},
}

Animals
*Hyperuricemia/genetics/metabolism
*CRISPR-Cas Systems/genetics
Disease Models, Animal
Mice
Humans
*5' Untranslated Regions/genetics
Gene Editing/methods
HEK293 Cells
*Gout/genetics/metabolism
*Metabolic Diseases/genetics
Gene Knockdown Techniques
Uric Acid/blood
Male
Mice, Inbred C57BL

@article {pmid41866453,
year = {2026},
author = {Wang, X and Xie, Y and Lin, Q and Xiong, Y and Liu, Y and Ge, S and Tan, Q and He, Z and Jiang, Y and Han, Q and Jin, S and Huang, P and Wang, Y and Guo, W and Ren, F and Gui, JF and Mei, J},
title = {Genetic deletion of miR-200a/200b increases growth and feed conversion efficiency in yellow catfish.},
journal = {Science China. Life sciences},
volume = {69},
number = {5},
pages = {1674-1687},
pmid = {41866453},
issn = {1869-1889},
mesh = {Animals ; *MicroRNAs/genetics ; *Catfishes/genetics/growth & development/metabolism ; Animal Feed ; *Gene Deletion ; Polymorphism, Single Nucleotide ; Energy Metabolism/genetics ; Genome-Wide Association Study ; Animals, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {The most effective approach for minimizing feed cost and maximizing animal production is the creation of breeding materials with simultaneous increases in growth and feed conversion efficiency (FCE). However, the key genes that regulate FCE are unknown. Here, we artificially selected specific strains of yellow catfish with simultaneous improvements in growth and FCE traits and then conducted a genome-wide association study to screen candidate SNPs and genes associated with these traits. A particular locus in the miR-200 cluster on chromosome 23 was identified, and the causal relationships between miR-200a/200b expression and growth/FCE were further validated. Genetic deletion of miR-200a/200b by CRISPR/Cas9 in yellow catfish significantly underpins phenotypic gains in growth and FCE by regulating genes involved in energy intake and energy metabolism without significantly affecting average feed intake or the expression of appetite-regulating genes. Several critical target genes of miR-200a/200b, such as stat5b and fasn, were identified via RNA-RNA pulldown and RNA-seq analyses, and stat5b-transgenic yellow catfish exhibited significantly increased growth and FCE. These findings highlight the pivotal role of the miR-200a/200b-stat5b signaling axis in controlling growth, metabolism, and FCE in yellow catfish, thus providing a strategy toward achieving more effective and sustainable animal agriculture by gene editing.},
}

Animals
*MicroRNAs/genetics
*Catfishes/genetics/growth & development/metabolism
Animal Feed
*Gene Deletion
Polymorphism, Single Nucleotide
Energy Metabolism/genetics
Genome-Wide Association Study
Animals, Genetically Modified
CRISPR-Cas Systems

@article {pmid42068455,
year = {2026},
author = {Chakraborty, A and Yu, ASL},
title = {Miniaturization of CRISPRa plasmids for efficient delivery into renal epithelial cells and Pkd1 transactivation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42068455},
issn = {1573-4978},
support = {24PRE1194472//American Heart Association/ ; U54 DK126126/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *TRPP Cation Channels/genetics/metabolism ; *Plasmids/genetics ; Mice ; Epithelial Cells/metabolism ; Kidney/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Transcriptional Activation/genetics ; Polycystic Kidney, Autosomal Dominant/genetics/therapy ; Genetic Vectors/genetics ; Gene Editing/methods ; },
abstract = {BACKGROUND: Autosomal Dominant Polycystic Kidney Disease is caused by loss-of-function mutations in PKD1 or PKD2 genes, leading to reduced polycystin protein levels. Increasing PKD1 expression via CRISPR activation (CRISPRa) represents a promising therapeutic strategy; however, delivery of large CRISPRa plasmids into renal epithelial cells, and particularly primary cells, remains inefficient due to size-related barriers. We aimed to enable Pkd1 transactivation by miniaturizing CRISPRa plasmids into ~ 6 kb vectors using a one-pot method to enhance cellular uptake in mouse kidney epithelial cells.

METHODS AND RESULTS: Using type IIS restriction enzymes, we excised the mammalian expression cassette from full-length large 9-11 kB plasmids. The excised cassette was engineered to have complimentary overhangs. Thermocycling with T4 DNA ligase promoted circularization of the excised cassette (forming ~ 6kB mini-CRISPRa vectors), and T5 exonuclease digestion removed residual backbone fragments. These mini vectors substantially enhanced nucleofection efficiency from 16.10% ± 0.53 to 54.17% ± 2.10 in Pkd1[RC/-] cells, and from 10.14% ± 1.40 to 31.27% ± 0.12 in primary Pkd1[RC/Cond]; Pkhd1[Cre+] cells. Functionally, the mini-CRISPRa plasmid (mdCas9-VPR) with Pkd1-targeting sgRNAs induced robust endogenous Pkd1 upregulation compared with non-targeting controls: a 4.1-fold increase in Pkd1[RC/-] cells (p < 0.001) and a 2.9-fold increase in primary cells (p < 0.001). Full-length plasmids produced no significant activation in either cell type.

CONCLUSIONS: Miniaturization of CRISPRa vectors with this one-pot approach overcomes delivery limitations in hard-to-transfect renal epithelial cells and enables efficient, functional Pkd1 activation, in vitro.},
}

Animals
*TRPP Cation Channels/genetics/metabolism
*Plasmids/genetics
Mice
Epithelial Cells/metabolism
Kidney/metabolism/cytology
*CRISPR-Cas Systems/genetics
Transcriptional Activation/genetics
Polycystic Kidney, Autosomal Dominant/genetics/therapy
Genetic Vectors/genetics
Gene Editing/methods

@article {pmid42069941,
year = {2026},
author = {Singh, A and Bhattacharjee, S and Singh, Y and Kostova, I},
title = {Parabiotics as Next-Generation Microbiome Therapeutics: Insights into Mechanisms, Evidence, and Therapeutic Potential.},
journal = {Current microbiology},
volume = {83},
number = {6},
pages = {},
pmid = {42069941},
issn = {1432-0991},
mesh = {Humans ; *Prebiotics/administration & dosage ; Animals ; *Gastrointestinal Microbiome/drug effects ; Probiotics ; *Microbiota ; },
abstract = {Parabiotics (also termed paraprobiotics) are defined as non-viable microbial cells or their components, including peptidoglycans, teichoic acids, surface proteins, that confer health benefits without requiring viability which distinguishes them from traditional probiotics. Their non-viable nature eliminates risks such as microbial translocation, bacteremia, and sepsis, making them suitable for vulnerable populations including immunocompromised, critically ill, paediatric and elderly individuals. In addition, parabiotic exhibit improved thermal stability, extended shelf life, and easier incorporation into functional foods, nutraceuticals, and pharmaceutical formulations without cold-chain requirements. Mechanistically, parabiotics retain immunomodulatory, anti-inflammatory and have barrier-enhancing activities through interactions with host pattern recognition receptors, including Toll-like receptors, modulation of cytokine responses, and reinforcement of gut epithelial integrity. Preclinical and clinical studies support their therapeutic potential such as in case of heat-killed Lactobacillus acidophilus LB (L. acidophilus) has shown efficiency in managing acute paediatric diarrhoea, while heat-inactivated Lacticaseibacillus paracasei PS23 (Lcb. paracasei) has demonstrated improvements in muscle strength and inflammatory markers, including reduced C-reactive protein and interleukin-6 and increased interlukin-10 in elderly individuals. Similarly, inactivated Lactiplantibacillus plantarum (Lpb. plantarum) and Bifidobacterium strains have been associated with benefits in irritable bowel syndrome, atopic dermatitis, respiratory infections, visceral fat reduction, and antibiotic-associated dysbiosis. Synergistic combinations with prebiotics, postbiotics and related bioactives further enhance therapeutic outcomes in inflammatory, metabolic and infectious conditions. Advances in metagenomics, next-generation sequencing, proteomics, metabolomics, CRISPR-Cas systems, and synthetic biology are accelerating strain characterization, functional evaluation, and scalable production. Despite ongoing challenges in standardization and regulated harmonization, parabiotics represent a safe and effective approach for microbiome-targeted interventions. This review synthesizes current evidence on their therapeutic applications, technological advancements, and translational potential, highlighting their role in precision health and next-generation functional nutrition.},
}

Humans
*Prebiotics/administration & dosage
Animals
*Gastrointestinal Microbiome/drug effects
Probiotics
*Microbiota

@article {pmid42070239,
year = {2026},
author = {Geerthana, S and Yogi, D and Kumar, A and Asokan, R and Suresh, K and Prabhakar, N and Rani, BU and Thangaraj, K and Paramasivam, M and Kaninika, V and Chiranth, RK and Shankarnarayan, AM and Pradeep, C and Manamohan, M},
title = {Towards CRISPR/Cas9 Genome Editing in Spodoptera frugiperda: A Proof-of-Concept Targeting Wing and Eye Color Genes.},
journal = {Archives of insect biochemistry and physiology},
volume = {122},
number = {1},
pages = {e70161},
doi = {10.1002/arch.70161},
pmid = {42070239},
issn = {1520-6327},
support = {//ICAR-IASRI for funding under the CABin programme/ ; },
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; *Spodoptera/genetics/growth & development ; Wings, Animal ; *Eye Color/genetics ; Pigmentation/genetics ; Tryptophan Oxygenase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; },
abstract = {Fall armyworm (Spodoptera frugiperda), a globally significant destructive lepidopteran invasive pest, has recently invaded Africa and Asia, threatening food security. Conventional method of management, including chemical insecticides, are often ineffective due to various reasons compelling the need to explore alternative strategies. In this regard, CRISPR/Cas9 based genome editing has emerged as a powerful tool for functional genomics in insects, enabling to introduce site-specific mutations for various purposes. In this study, we applied multiplex CRISPR/Cas9 ribonucleoprotein (RNP) injections to disrupt two key genes in S. frugiperda: the spalt (Sfspalt), which regulates wing patterning, and tryptophan 2,3-dioxygenase (Sfto/vermillion) gene, involved in eye pigmentation. Microinjection of sgRNA/Cas9 ribonucleoprotein complex into freshly laid eggs resulted in distinct phenotypic alterations, including altered wing pigmentation and modified eyespot patterns, as well as golden-yellow eye color mutants. Genotyping and ICE analysis confirmed the presence of frameshift mutations in the target loci, supporting the phenotypic changes. Notably, while mutations were detected, only a single individual was confirmed to carry mutations in both genes simultaneously. These results demonstrate the technical feasibility of multiplex CRISPR/Cas9 editing in S. frugiperda, but also reveal a low frequency of confirmed events under the present experimental conditions. Therefore, this study is considered a proof of concept establishing a preliminary workflow in multiplex platform. The findings provide foundational insights for further optimization of genome editing strategies targeting sex related genes in this agriculturally important pest.},
}

Animals
*Gene Editing
*CRISPR-Cas Systems
*Spodoptera/genetics/growth & development
Wings, Animal
*Eye Color/genetics
Pigmentation/genetics
Tryptophan Oxygenase/genetics/metabolism
*Insect Proteins/genetics/metabolism

@article {pmid41870705,
year = {2026},
author = {Khoshraftar, SH and Alirezae, P and Kiani Darabi, AH and Hadi, S and Gholami, A and Amirfiroozi, A and Pourseif, MM and Mansoori-Derakhshan, S},
title = {The role of circular RNAs as miRNA sponges in the mechanisms and therapeutic potential of triple negative breast cancer.},
journal = {Discover oncology},
volume = {17},
number = {1},
pages = {},
pmid = {41870705},
issn = {2730-6011},
support = {73169//Tabriz University of Medical Sciences/ ; },
abstract = {Triple-negative breast cancer (TNBC) is an aggressive subtype of breast carcinoma which lacks estrogen receptors, progesterone receptors and HER2 along with limited therapeutic options mainly based on chemotherapy. In this review, we outline the emerging function of circRNAs as key regulators in TNBC pathogenesis. CircRNAs are endogenous non-coding RNAs with a closed-loop structure, in contrast to the linear form. In TNBC cells, the underlying molecular mechanism mainly relies on their functions as a competitive sponge of miRNAs, which can absorb or bind to microRNAs (miRNAs) and hence regulate the expression of target genes. Such sponging can result in the activation of oncogenes or repression of tumor suppressor genes, which eventually affect cellular proliferation, apoptosis, and drug sensitivity. Crucial mechanisms include certain circRNAs, such as circEPSTI1, circRAD18, and hsacirc0000199 that enhance tumorigenesis and resistance to chemotherapy by targeting tumor-suppressor miRNAs and activation of oncogenic pathways (e.g., PI3K/Akt/mTOR pathway or Wnt/β-catenin). The potential clinical implications and dysregulation of circRNA-miRNA axes are highlighted, indicating that these may constitute promising diagnostic or prognostic markers by their stability in biofluids. Additionally, this review outlines the innovative treatment approaches regarding these interactions which have recently been addressed and described, novel methods include ASOs therapy, CRISPR/Cas system and nanoplatforms that may help to get over current therapeutic drawbacks in treating TNBC patients.},
}

@article {pmid40356240,
year = {2025},
author = {Coveney, CR and Maridas, D and Chen, H and Muthuirulan, P and Liu, Z and Jagoda, E and Yarlagadda, S and Movahhedi, M and Proffen, B and Dashtdar, B and Aghaalikhani, M and Richard, D and Rosen, V and Kiapour, AM and Capellini, TD},
title = {Complex Regulatory Interactions at GDF5 Shape Joint Morphology and Osteoarthritis Disease Risk.},
journal = {Arthritis & rheumatology (Hoboken, N.J.)},
volume = {77},
number = {11},
pages = {1488-1502},
pmid = {40356240},
issn = {2326-5205},
support = {//Institutional Centers for Clinical and Translational Research at Boston Children's Hospital/ ; R01 AR070139/AR/NIAMS NIH HHS/United States ; P30 AR075042/AR/NIAMS NIH HHS/United States ; //Harvard University Dean's Competitive Fund/ ; 1R01AR070139/NH/NIH HHS/United States ; 1UM1TR004408-01/NH/NIH HHS/United States ; //Harvard Clinical and Translational Science Center/ ; //The Children's Orthopaedic Surgery Foundation/ ; UM1 TR004408/TR/NCATS NIH HHS/United States ; //Harvard University Milton Fund/ ; P30 AR075042/NH/NIH HHS/United States ; 1R01AR070139/NH/NIH HHS/United States ; 1UM1TR004408-01/NH/NIH HHS/United States ; P30 AR075042/NH/NIH HHS/United States ; },
mesh = {*Osteoarthritis/genetics/metabolism/pathology ; *Growth Differentiation Factor 5/genetics/metabolism ; Disease Models, Animal ; Gene Expression Regulation ; Humans ; Female ; Animals ; Mice/embryology/metabolism ; Male ; Cartilage, Articular/metabolism/pathology ; Joints/metabolism ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: The objective of this study was to reveal causal-level osteoarthritis (OA) disease biology by targeting regulatory interactions at GDF5.

METHODS: By investigating different GDF5 regulatory regions (R2, R3-R5, R7-R9, R18-R20, GROW1), we explored their functional impacts on gene expression and joint morphology in vivo and in vitro. We additionally modeled OA variants in said enhancers in in vitro and in vivo mouse models for expression and disease effects.

RESULTS: For all regulatory regions, we found evidence of activation and repression between or within said regions that impacted patterns of joint-specific expression. Examples are as follows: (1) the R4 enhancer, although considered to be activating, has dual roles repressing expression in adjacent tissues and sites, and (2) growth plate-specific expression patterns by the GROW1 regulatory region are confined by adjacent sequences to restrict its expression to the perichondrium. We next targeted different regions and variants in vivo. Testing the R2de region resulted in ~40% reduction in Gdf5 expression and joint morphology changes but no increase in OA risk; likewise, modeling the most cited OA risk variant (rs143384) in mice had no impact on expression, joint morphology, or disease. However, we identified epistatic interactions between this rs143384 risk variant and downstream disease risk variants lying within regulatory regions subject to repression, which compound to impact expression.

CONCLUSION: These findings, at the best studied OA locus to date, serve as lessons on the nature of how gene regulatory interactions and local epistasis work in the etiology of OA disease risk, and that assessment of individual variants of high genome-wide association study significance need not alone be considered causal.},
}

*Osteoarthritis/genetics/metabolism/pathology
*Growth Differentiation Factor 5/genetics/metabolism
Disease Models, Animal
Gene Expression Regulation
Humans
Female
Animals
Mice/embryology/metabolism
Male
Cartilage, Articular/metabolism/pathology
Joints/metabolism
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid41394860,
year = {2025},
author = {Andersch, L and Grunewald, L and Stecklum, M and Klironomos, F and Haase, K and Hollek, V and Lam, T and Jung, BA and Winkler, A and Schwiebert, S and Astrahantseff, K and Launspach, M and Jens, M and Henssen, A and Kloke, L and Blüthgen, N and Eggert, A and Schulte, JH and Anders, K and Künkele, A},
title = {Investigating genetic modifications to enhance L1CAM-CAR T cell migration in solid tumors in a 3D bioprinted neuroblastoma model.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1677361},
pmid = {41394860},
issn = {1664-3224},
mesh = {*Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Neuroblastoma/immunology/pathology/therapy ; Bioprinting ; *Immunotherapy, Adoptive/methods ; *Cell Movement/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; Receptors, Chimeric Antigen/immunology/metabolism ; Neural Cell Adhesion Molecule L1/immunology/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Membrane Glycoproteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/deficiency/genetics/metabolism ; Xenograft Model Antitumor Assays ; Single-Cell Gene Expression Analysis ; Humans ; Female ; Animals ; Mice ; Cell Line, Tumor ; Coculture Techniques ; Gene Expression Regulation/immunology ; },
abstract = {INTRODUCTION: Effective CAR T cell infiltration into solid tumors remains a major barrier to therapy success. Despite their clinical potential, few studies have evaluated phenotypes of CAR T cells successfully invading the tumor mass following infusion. Phenotypic information would enrich our understanding of the mechanisms governing CAR T cell migration into solid tumors. Here we implemented an in vitro strategy to identify genes driving L1CAM-CAR T cell migration into a 3D tumor mass.

METHODS: L1CAM-CAR T cells were separated into 2 groups by their capability to infiltrate (or not) a 3D bioprinted neuroblastoma model. Single-cell and bulk RNA sequencing was performed, and infiltrating CAR T cells were compared to noninfiltrating cells to seek genetic drivers of CAR T cell migration. CRISPR/Cas9 technology was used to generate modified L1CAM-CAR T cells.

RESULTS: Tumor-infiltrating L1CAM-CAR T cells expressed lower levels of the selectin P ligand (SELPLG) glycoprotein and higher levels of the T cell-specific adaptor protein, SH2D2A. Functional characterization of L1CAM-CAR T cells genetically modified to enforce these characteristics demonstrated that neither trait negatively impacted L1CAM-CAR T cell cytotoxicity, activation and cytokine release upon coculture with neuroblastoma target cells. Transgenic SH2D2A expression did not improve CAR T cell migration in an endothelial transmembrane assay. SELPLG knockout benefited CAR T cell in vitro trans-endothelial migration, but did not enhance anti-tumor efficacy in an immunodeficient mouse model.

DISCUSSION: Our findings reveal a key limitation of murine xenograft models, which are widely used as the gold standard for preclinical CAR T cell testing. The lack of conservation between the human and murine SELPLG proteins likely accounts for the discrepancy between enhanced in vitro migration of SELPLG-deficient L1CAM-CAR T cells and their lack of improved efficacy in the mouse model. This underscores the need for more predictive human-relevant models to better preclinically evaluate CAR T cell function.},
}

*Lymphocytes, Tumor-Infiltrating/immunology/metabolism
*Neuroblastoma/immunology/pathology/therapy
Bioprinting
*Immunotherapy, Adoptive/methods
*Cell Movement/genetics/immunology
*T-Lymphocytes/immunology/metabolism
Receptors, Chimeric Antigen/immunology/metabolism
Neural Cell Adhesion Molecule L1/immunology/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Membrane Glycoproteins/genetics/metabolism
Adaptor Proteins, Signal Transducing/deficiency/genetics/metabolism
Xenograft Model Antitumor Assays
Single-Cell Gene Expression Analysis
Humans
Female
Animals
Mice
Cell Line, Tumor
Coculture Techniques
Gene Expression Regulation/immunology

@article {pmid41877120,
year = {2026},
author = {Wang, Y and Cao, M and Hu, M and Xu, H and Du, Y and Sun, C and Kong, L and Luo, Y and Liu, X and Yang, J and Liang, B},
title = {Systematic engineering of Escherichia coli for biosynthesis of 3-hydroxypropionic acid from glucose and malonate.},
journal = {BMC biotechnology},
volume = {26},
number = {1},
pages = {},
pmid = {41877120},
issn = {1472-6750},
support = {22278233//National Natural Science Foundation of China/ ; 22378222//National Natural Science Foundation of China/ ; ZR2025MS127//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Malonates/metabolism ; *Metabolic Engineering/methods ; *Glucose/metabolism ; *Lactic Acid/analogs & derivatives/biosynthesis/metabolism ; CRISPR-Cas Systems ; Fermentation ; Malonyl Coenzyme A/metabolism ; },
abstract = {BACKGROUND: 3-Hydroxypropionic acid (3-HP) is a promising C3 platform chemical with wide industrial applications. However, its microbial production remains limited by insufficient intracellular malonyl-CoA availability and metabolic imbalance.

RESULT: In this study, we systematically engineered Escherichia coli for enhanced 3-HP biosynthesis. The malonate assimilation genes (matB, smatPQM) and 3-HP biosynthesis gene (mcr) were chromosomally integrated using CRISPR/Cas9, resulting in a plasmid-free, antibiotic-free strain (WYY04) that produced 21.97 mM 3-HP, 0.51-fold higher than the plasmid-based system. Further improvement was achieved by CRISPRi-mediated repression of fatty acid biosynthesis genes (fabD, fabF), increasing 3-HP titer by 66%. Introduction of a malonyl-CoA-responsive FapR/fapO biosensor enabled dynamic regulation of mcr expression, enhancing 3-HP production by 59%. Through all these above engineering, the 3-HP production of the strain WYY19 increased by 2.29 times compared to that of the plasmid-expressing system. Under optimized fermentation conditions, the final engineered strain WYY19 produced 42.22 g/L 3-HP with the specific productivity of 0.69 g/g and 0.46 g/L/h from glucose and malonate in fed-batch bioreactor.

CONCLUSIONS: This study demonstrates a robust, genetically stable, and scalable microbial platform for 3-HP biosynthesis.},
}

*Escherichia coli/genetics/metabolism
*Malonates/metabolism
*Metabolic Engineering/methods
*Glucose/metabolism
*Lactic Acid/analogs & derivatives/biosynthesis/metabolism
CRISPR-Cas Systems
Fermentation
Malonyl Coenzyme A/metabolism

@article {pmid41945404,
year = {2026},
author = {Yu, F and Zhang, D and Peng, C and Qin, H and Xu, F and Zhang, Y and Pan, Z and Xiao, P and Li, N},
title = {Development of a rapid and sensitive single-tube RAA-CRISPR/Cas12a assay for monkeypox virus detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3312-3320},
doi = {10.1039/d6ay00065g},
pmid = {41945404},
issn = {1759-9679},
mesh = {*Monkeypox virus/isolation & purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Sensitivity and Specificity ; Limit of Detection ; Recombinases/metabolism ; },
abstract = {Monkeypox (mpox) is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks historically concentrated in West and Central Africa. The current global outbreak of MPXV highlights the urgent need for an efficient detection system. This study presents a novel single-tube recombinase-aided amplification (RAA)-CRISPR/Cas12a assay for the rapid, sensitive, and specific detection of MPXV. Optimized to achieve a fluorescence-based sensitivity as low as 0.5 copies per µL within 35 min at 37 °C, the system demonstrates excellent specificity, accurately distinguishing MPXV from other viruses, including vaccinia virus, with no cross-reactivity observed. Additionally, a lateral flow assay (LFA) format was developed, enabling visual detection of concentrations as low as 5 copies per µL within 40 min. Validated with simulated clinical samples, the assay achieved 100% accuracy in distinguishing positives from negatives. This integrated single-tube approach eliminates the need for costly thermal cyclers, simplifying the detection process, reducing contamination risks, and delivering reliable results in a short time, making it ideal for point-of-care testing (POCT) in resource-limited settings. Furthermore, the RAA-CRISPR/Cas12a platform offers significant cost savings by requiring fewer reagents for virus detection. The extremely low template volume requirement of just 1 µL maximizes detection efficiency. This configuration allows for repeated testing without compromising result integrity, further enhancing the utility of this approach for surveillance and outbreak control, particularly in low-resource environments.},
}

*Monkeypox virus/isolation & purification/genetics
*Mpox, Monkeypox/diagnosis/virology
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
Sensitivity and Specificity
Limit of Detection
Recombinases/metabolism

@article {pmid41960606,
year = {2026},
author = {Zhang, X and Nie, X and Yu, W and Du, G and Liu, S and Song, Y},
title = {Catalytic hairpin assembly-assisted split-T7 promoter-regulated CRISPR/Cas12a system for the sensitive analysis of microRNAs associated with coronary heart disease.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3303-3311},
doi = {10.1039/d6ay00524a},
pmid = {41960606},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Coronary Disease/genetics/diagnosis ; *Promoter Regions, Genetic/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) have emerged as promising non-invasive biomarkers for coronary heart disease (CHD); however, their accurate quantification remains challenging due to their short length, low abundance, and high sequence homology. Herein, we report a highly sensitive and specific biosensing platform by integrating catalytic hairpin assembly (CHA) with a split-T7 promoter-regulated CRISPR/Cas12a system. The key innovation lies in the split-T7 promoter architecture, which enables target-responsive reconstitution of a functional T7 promoter, driving robust in vitro transcription to generate abundant crRNA transcripts eliminating the need for pre-synthesized exogenous crRNA. This strategy achieves triple cascade amplification through three synergistic modules: CHA-mediated target recycling ensures efficient signal initiation; split-T7 promoter-driven transcription provides substantial signal enrichment; and CRISPR/Cas12a-based trans-cleavage delivers highly specific fluorescence readout. By eliminating pre-synthesized crRNA, the platform significantly reduces assay cost and complexity while maintaining excellent amplification efficiency. The method achieves a limit of detection as low as 38.9 aM for miRNA-155 within 90 min, exhibits single-base mismatch discrimination capability, and performs reliably in clinical specimens from CHD patients. With its modular design and robust performance, this cascade amplification platform offers a versatile and cost-effective tool for miRNA analysis, holding great promise for cardiovascular disease diagnosis and broader molecular diagnostics.},
}

*MicroRNAs/genetics/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
*Coronary Disease/genetics/diagnosis
*Promoter Regions, Genetic/genetics
*Biosensing Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42064640,
year = {2026},
author = {Pandey, H and Sharma, A and Misra, V and Mall, AK and Ceasar, SA},
title = {Pre-validation strategies for CRISPR/Cas-based genome editing in plants: a critical analysis of in vitro RNP cleavage assays.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {4},
pages = {677-691},
pmid = {42064640},
issn = {0971-5894},
abstract = {The advent of CRISPR/Cas-based genome editing has revolutionized crop improvement. However, the genome editing success rate remains a major challenge in many crops, especially those with challenging transformation protocols. We critically evaluate the integration of in vitro cleavage assays using naked target DNA and guide RNA-Cas9 nuclease (gRNA-Cas9) ribonucleoprotein (RNP) complexes as a pre-transformation validation step in genome editing workflows. We also compare other pre-validation methods with in vitro cleavage assays and present their advantages and limitations. In vitro assays can help directly confirm target cleavage and biochemically validate gRNA specificity. This strategy may facilitate the functional screening of gRNAs for plants with challenging and low transformation efficiency. In vitro assays can also reduce the unnecessary waste of resources and time associated with intensive transformation processes using non-specific gRNAs. Researchers can prioritize effective constructs based on the cleavage efficiency and specificity of the gRNAs. However, this assay may not guarantee simulation of the natural cellular environment for in vivo editing. We also present this pre-validation approach, which is particularly helpful for polyploid crops like wheat and cotton. In vitro cleavage assays offer a reliable pre-transformation screening step to identify highly active and specific gRNAs, thereby reducing resource-intensive transformation attempts. Future studies should integrate in vitro assays with advanced computational and in vivo validation tools to create a more predictive and efficient gRNA selection pipeline.},
}

@article {pmid42065027,
year = {2026},
author = {Ezeobiora, CE and Igbokwe, NH and Amin, DH and Mendie, UE},
title = {Rare endophytic actinobacteria from nigeria harbor unique biosynthetic gene clusters with novel antibiotic potential.},
journal = {3 Biotech},
volume = {16},
number = {5},
pages = {174},
pmid = {42065027},
issn = {2190-572X},
abstract = {UNLABELLED: Actinobacteria are prolific producers of specialized metabolites, including antibiotics; however, much of their biosynthetic potential remains unexplored, particularly within rare genera. This study presents the first genomic insights into the biosynthetic capacity of two rare endophytic actinobacteria, Saccharomonospora xinjiangensis strain PNSac2 and Saccharopolyspora cebuensis strain PGLac3, isolated from medicinal plants in Nigeria. The strains were characterized using morphological analysis, molecular sequencing, phylogenetic inference, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH). Whole-genome sequencing revealed that PNSac2 possesses a 4.7 Mb genome with 45 tRNA genes, 3 rRNA operons, and 4,541 coding sequences (CDSs), while PGLac3 harbors a 6.4 Mb genome comprising 48 tRNA genes, 4 rRNA operons, and 6,372 CDSs. Genome mining using antiSMASH identified 24 biosynthetic gene clusters (BGCs) in PNSac2 and 28 in PGLac3, including clusters encoding polyketides, nonribosomal peptides, siderophores, terpenes, and ribosomally synthesized and post-translationally modified peptides (RiPPs). Many BGCs showed low similarity to known clusters, indicating a strong potential for novel metabolite discovery. Notably, PNSac2 encoded BGCs related to bleomycin, oxalomycin, desertomycin, and ossamycin, while PGLac3 harbored predicted arylpolyene, lanthipeptide, and a unique lassopeptide cluster. Comparative genomics revealed conserved synteny with related species alongside strain-specific BGCs, and phylogenomic analysis confirmed their taxonomic placement. Overall, these findings highlight the untapped biosynthetic diversity of rare Nigerian endophytic actinobacteria and underscore their promise as sources of novel antimicrobial compounds. Targeted genome engineering approaches, including CRISPR-Cas-based strategies, may further enable the activation and exploitation of cryptic biosynthetic pathways in these strains.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-026-04781-4.},
}

@article {pmid42067284,
year = {2026},
author = {Zhang, X and Tian, C and Wang, M and Jia, H and Li, X and Tian, G},
title = {Performance enhancement of CRISPR-Cas system based on improved guide RNA: a review.},
journal = {Analytica chimica acta},
volume = {1406},
number = {},
pages = {345472},
doi = {10.1016/j.aca.2026.345472},
pmid = {42067284},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Biosensing Techniques ; },
abstract = {BACKGROUND: CRISPR-Cas technology has emerged as a transformative tool with widespread applications in gene editing and biosensing research; nevertheless, it is plagued by a suite of performance-related bottlenecks, including suboptimal targeting efficiency, undesirable off-target effects, insufficient sensitivity and recognition specificity, restricted target scope, limited multiplexing capacity, incompatible reaction systems, and compromised stability. As gRNA optimization has emerged as a core strategy to address these bottlenecks, there is an urgent need to consolidate recent breakthroughs in this rapidly advancing field. Existing literature lacks a comprehensive, focused synthesis of how gRNA optimization mitigates these key limitations, alongside an analysis of current challenges and future directions.

RESULTS: Herein, this review comprehensively summarizes recent breakthroughs in augmenting CRISPR-Cas system performance through guide RNA (gRNA) optimization, and further dissects the current challenges, future prospects, and promising research directions in this rapidly advancing field.

SIGNIFICANCE: It is timely to guide researchers in overcoming CRISPR-Cas performance barriers and accelerating its applications in gene editing and biosensing.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Humans
Biosensing Techniques

@article {pmid42067668,
year = {2026},
author = {Wu, X and Lam, WH and Zhao, Z and Cao, Y and Lin, H and Feng, X and Zhai, Y and Hsing, IM},
title = {DNA-guided CRISPR-Cas12a effectors for programmable RNA recognition and cleavage.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42067668},
issn = {1546-1696},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 6107-20G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 6107-20G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C6053-25G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C7035-23GF//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C6053-25G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C7035-23GF//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 325014//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.},
}

@article {pmid41520142,
year = {2026},
author = {Brown, A and Strickland, LM and Erman, EN and Pirozzi, CJ and Low, JT and Diplas, BH and Gibson, E and Shobande, M and Khambati, T and Krylova, M and Liu, H and McLendon, RE and Reitman, ZJ and Keir, ST and Zou, L and Ashley, DM and Waitkus, MS},
title = {SMARCAL1 is a targetable synthetic lethal therapeutic vulnerability in ATRX-deficient gliomas that use alternative lengthening of telomeres.},
journal = {Neuro-oncology},
volume = {28},
number = {4},
pages = {895-910},
pmid = {41520142},
issn = {1523-5866},
support = {P50 CA108786/CA/NCI NIH HHS/United States ; F32 CA298007/CA/NCI NIH HHS/United States ; P50 CA190991/CA/NCI NIH HHS/United States ; K22 CA258965/CA/NCI NIH HHS/United States ; R01 CA091438/CA/NCI NIH HHS/United States ; P30 CA014236/CA/NCI NIH HHS/United States ; T32 GM145449/GM/NIGMS NIH HHS/United States ; //The Preston Robert Tisch Brain Tumor Center/ ; //Uncle Kory Foundation Seed/ ; SPORE 5P50CA190991//Duke Brain Tumor/ ; 1K22CA258965-01A1/GF/NIH HHS/United States ; 1F32CA298007-01/GF/NIH HHS/United States ; },
mesh = {Humans ; *X-linked Nuclear Protein/deficiency/genetics ; Animals ; *Glioma/genetics/pathology/metabolism ; *Telomere Homeostasis ; Mice ; *Brain Neoplasms/genetics/pathology/metabolism ; Xenograft Model Antitumor Assays ; *Telomere/genetics ; *DNA Helicases/genetics ; Tumor Cells, Cultured ; *Synthetic Lethal Mutations ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {BACKGROUND: Approximately 10% of cancers achieve replicative immortality through a telomerase-independent mechanism of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). ALT is particularly prevalent in certain subtypes of malignant gliomas, such as IDH-mutant astrocytoma and pediatric glioblastoma, and frequently co-occurs with ATRX (ATRX chromatin remodeler) inactivating mutations. Although ALT is an adaptive mechanism through which cancer cells achieve proliferative immortality, the elevated levels of replication stress observed in ALT tumors constitute a potential therapeutic vulnerability.

METHODS: Leveraging CRISPR/Cas9 screening data from the Cancer Dependency Mapping Project, coupled with patient-derived cell lines and xenografts, we identified SMARCAL1 as a novel synthetic lethal vulnerability in ATRX-deficient glioma models that engage ALT. Using complementary molecular assays for DNA damage, telomere maintenance, and telomeric replication stress, we define the mechanisms underlying cytotoxicity induced by SMARCAL1 depletion in ALT-positive glioma cells.

RESULTS: Our data demonstrate the annealing helicase SMARCAL1 is a highly specific synthetical lethal vulnerability in cancers that use ALT. SMARCAL1 localizes to ALT-associated PML (Promyelocytic leukemia protein) bodies in ALT-positive glioma cell lines, including IDH-mutant astrocytomas. SMARCAL1 depletion, via doxycycline-induced RNAi, led to a hyperactivation of the ALT phenotype, high levels of DNA double-strand breaks in G2 phase, and cell death via mitotic catastrophe. In mice bearing intracranial xenografts derived from high-grade IDH-mutant astrocytoma, inducible SMARCAL1 depletion prolonged animal survival.

CONCLUSIONS: Our findings demonstrate that the molecular processes orchestrating ALT-mediated telomere maintenance constitute a targetable synthetic lethal vulnerability that can be exploited by SMARCAL1 inhibition, thus supporting the future development of small molecule inhibitors of SMARCAL1 as anti-cancer therapeutics.},
}

Humans
*X-linked Nuclear Protein/deficiency/genetics
Animals
*Glioma/genetics/pathology/metabolism
*Telomere Homeostasis
Mice
*Brain Neoplasms/genetics/pathology/metabolism
Xenograft Model Antitumor Assays
*Telomere/genetics
*DNA Helicases/genetics
Tumor Cells, Cultured
*Synthetic Lethal Mutations
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid41851324,
year = {2026},
author = {Zaboroski-Silva, I and da Silva Brandão, E and de Freitas Brenha, B and Landry, C and Carrara, J and Ferreira, RS and Vaz, IM and Jamur, VR and Schaffer, AE and Miranda, HC and Shigunov, P},
title = {Impact of the CYFIP2 R87C variant in a human neuronal model in vitro.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41851324},
issn = {2045-2322},
support = {PDPG-FAP 88887.629870/2021-0//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; R01 NS121374/NS/NINDS NIH HHS/United States ; 15/2019-PROEP/ICC-442324/2019-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; PEP ICC-007-FEX-23-2-1-36//Financiadora de Estudos e Projetos/ ; R01 NS121374/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Neurons/metabolism/cytology ; *Neural Stem Cells/metabolism/cytology ; Organoids/metabolism/cytology ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Cell Differentiation/genetics ; Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Mutation ; Cell Line ; Neurogenesis/genetics ; },
abstract = {Mutations in the CYFIP2 gene, particularly the R87C variant, are associated with severe epileptic encephalopathy, and present challenges for therapeutic development. This study utilized CRISPR/Cas9-edited human pluripotent stem cell (hPSC) lines to investigate the impact of R87C variant on neuronal morphology and function. hPSCs were differentiated into neural progenitor cells (NPCs), cortical neurons (CNs), and cortical organoids. Phenotypic characterization included immunofluorescence, scanning electron microscopy (SEM), high-throughput scanning (HTS), multi-electrode array (MEA) recordings, and Western blotting. Edited hPSC lines maintained pluripotency, and neurogenic differentiation yielded NPCs and CNs without significant differences in neural progenitor marker expression. However, mutated NPCs exhibited reduced motility in cell tracking assays, and SEM revealed altered cell morphology, suggesting an impact on lamellipodia formation. While both mutant and wild-type CNs expressed appropriate neuronal and glial markers and showed similar electrophysiological properties, R87C/R87C cortical organoids displayed decreased CYFIP2 protein levels and, by day 30, showed increased size alongside an absence of SOX2 + cells, suggesting premature depletion of the progenitor pool. These findings highlight a marked divergence between 2D and 3D models, with organoids revealing neurodevelopmental abnormalities not evident in monolayer cultures. Together, our results suggest that the CYFIP2 R87C variant impacts NPC cytoskeletal dynamics and early cortical development, warranting further investigation into its role in epileptic encephalopathy.},
}

Humans
*Neurons/metabolism/cytology
*Neural Stem Cells/metabolism/cytology
Organoids/metabolism/cytology
*Adaptor Proteins, Signal Transducing/genetics/metabolism
Cell Differentiation/genetics
Pluripotent Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Mutation
Cell Line
Neurogenesis/genetics

@article {pmid42044121,
year = {2026},
author = {Varderesian, HV and Utaegbulam, JN and Brown, HE and Ramirez, B and Velcani, M and Ryder, SP},
title = {The pos-1 3' untranslated region governs germline specification and proliferation to ensure reproductive robustness.},
journal = {PLoS genetics},
volume = {22},
number = {4},
pages = {e1012129},
pmid = {42044121},
issn = {1553-7404},
mesh = {Animals ; *3' Untranslated Regions/genetics ; Caenorhabditis elegans/genetics ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; Female ; Male ; Germ Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Proliferation/genetics ; Zygote/metabolism/growth & development ; Reproduction/genetics ; Fertilization/genetics ; Oocytes/metabolism ; Fertility/genetics ; CRISPR-Cas Systems ; },
abstract = {During fertilization, haploid gametes combine to form a zygote. The male (sperm) and female (oocyte) gametes contribute a similar amount of DNA, but the oocyte contributes nearly all the cytoplasm. Oocytes are loaded with maternal mRNAs thought to be essential for embryonic patterning after fertilization. A conserved suite of RNA-binding proteins (RBPs) regulates the spatiotemporal translation and stability of maternal mRNAs. POS-1 is a CCCH-type tandem zinc finger RBP expressed in fertilized Caenorhabditis elegans zygotes from maternally supplied mRNA. POS-1 accumulates in the posterior of the embryo where it promotes posterior cell fate. Here, we show that the pos-1 3' untranslated region (UTR) is essential for POS-1 patterning and contributes to maximal reproductive fecundity. We engineered a pos-1 mutant where most of the endogenous pos-1 3'UTR was removed using CRISPR genome editing. Our results show that the 3'UTR represses POS-1 expression in the maternal germline but increases POS-1 protein levels in embryos after fertilization. In a wild-type background, POS-1 repression via the 3'UTR has little impact on fertility. In a sensitized background, the deletion mutant has a complex pleiotropic phenotype where most adult homozygous progeny lack either one or both gonad arms. Most phenotypes become more penetrant at elevated temperature. Together, our results support an emerging model where the 3'UTRs of maternal transcripts, rather than being essential, contribute to reproductive robustness during stress.},
}

Animals
*3' Untranslated Regions/genetics
Caenorhabditis elegans/genetics
*Caenorhabditis elegans Proteins/genetics/metabolism
*RNA-Binding Proteins/genetics/metabolism
Female
Male
Germ Cells/metabolism
Gene Expression Regulation, Developmental
Cell Proliferation/genetics
Zygote/metabolism/growth & development
Reproduction/genetics
Fertilization/genetics
Oocytes/metabolism
Fertility/genetics
CRISPR-Cas Systems

@article {pmid42057199,
year = {2026},
author = {Hatem, H and Mysara, M and Ramadan, R},
title = {Trends of nucleic acid - based point-of-care diagnostics for infectious diseases.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {},
pmid = {42057199},
issn = {1754-1611},
abstract = {UNLABELLED: The global emergence and spread of infectious diseases highlights the importance of having an easily accessible, decentralized testing modality that is adept at quickly identifying the nucleic acids present in a Point-of-Care (PoC) setting. PoC-based nucleic acid diagnostics encompass a broad range of platforms, such as lateral flow, biochips, and biosensors, whose primary goal has always been to offer sensitive, selective, and economically viable testing beyond conventional laboratory facilities. The current review illustrates a comprehensive overview of PoC-based nucleic acid diagnostics for infectious diseases, as it is divided into three major operational steps: nucleic acid extraction, amplification, and detection. Firstly, it describes strategies that could effectively work as a PoC, such as magnetic bead-based, paper-based, and integrated microfluidic approaches, while further focusing on their ability to remain simplified, rugged, and equipment-free. Secondly, it summarizes key amplification methods, such as Polymerase Chain Reaction (PCR), Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), and Loop-Mediated Isothermal Amplification (LAMP), as they have all been modified to effectively work under more rapid, low-power, and portable conditions. Lastly, it introduces all presently known detection platforms, starting from simpler colorimetric and fluorescent assays present in lateral flow or device platforms, to innovative and advanced biosensors like those exploiting CRISPR/Cas systems and toehold switch principles, as these detectively offer highly sensitive and highly programmable nucleic acid recognition.

GRAPHICAL ABSTRACT: [Image: see text]},
}

@article {pmid42058176,
year = {2026},
author = {McNiven, C and Carnielli Trindade, JB and Geoghegan, V and Faria, JRC and Mottram, JC},
title = {CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1788564},
pmid = {42058176},
issn = {2235-2988},
mesh = {*Leishmania mexicana/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Kinetochores/metabolism ; *Protozoan Proteins/genetics/metabolism ; Phosphorylation ; *Codon/genetics ; },
abstract = {Leishmania mexicana, like other trypanosomatids, possess a unique kinetochore-the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160 bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.},
}

*Leishmania mexicana/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Kinetochores/metabolism
*Protozoan Proteins/genetics/metabolism
Phosphorylation
*Codon/genetics

@article {pmid42059198,
year = {2026},
author = {Hellmer, H and Mayer, T and Bauersachs, L and Simmel, FC},
title = {Operating CRISPR/Cas12a in a complex nucleic acid sequence background.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag390},
pmid = {42059198},
issn = {1362-4962},
support = {SI761/5-1//Deutsche Forschungsgemeinschaft/ ; 453249455//Deutsche Forschungsgemeinschaft/ ; CRC392 TP A5//Deutsche Forschungsgemeinschaft/ ; 521256690-TPA5//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA/metabolism/genetics/chemistry ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Gene Editing/methods ; Base Composition ; Base Sequence ; Humans ; },
abstract = {Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA (gRNA) design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a-a Class 2, Type V effector-and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the gRNA seed region significantly affect kinetic inhibition by background polynucleotides. gRNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. A gRNA with high uracil content in the seed region exhibited particularly strong inhibition in the presence of a dsDNA background. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
DNA/metabolism/genetics/chemistry
Kinetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
Gene Editing/methods
Base Composition
Base Sequence
Humans

@article {pmid42060714,
year = {2026},
author = {Jung, M and Wen, Z and Humbert, S and Lu, F and DeLeon, A and Marshall, L and Hastings, C and Cartwright, H and Thilges, K and Wang, N and Breckenridge, K and Wu, E and Ryan, L and Fengler, K and Simcox, K and Thatcher, S and Llaca, V and Woollums, G and Sander, J and Xu, D and Beatty, M and Brink, K and Fedorova, M and Jones, M and Ohlson, E and Suresh, LM and Beyene, Y and Olsen, M and Ogugo, V and Alakonya, A and Murithi, A and Mugo, S and Karanja, J and Boddupalli, P and Pixley, K and Albertsen, M and Jones, T and Meeley, R and Gutterson, N and Mazur, B and Dhugga, KS},
title = {Targeted knockout of a host peroxisomal peptidase confers field resistance to maize lethal necrosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {18},
pages = {e2535202123},
doi = {10.1073/pnas.2535202123},
pmid = {42060714},
issn = {1091-6490},
support = {OPP1193833//Bill and Melinda Gates Foundation (GF)/ ; INV-007833/GATES/Gates Foundation/United States ; },
mesh = {*Plant Diseases/virology/genetics ; *Zea mays/virology/genetics ; *Disease Resistance/genetics ; *Peroxisomes/enzymology/genetics ; Potyvirus/pathogenicity ; Gene Knockout Techniques ; *Peptide Hydrolases/genetics/metabolism ; Quantitative Trait Loci/genetics ; Tombusviridae/pathogenicity ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; },
abstract = {Maize lethal necrosis (MLN) is a severe disease caused by the combined infection of maize chlorotic mottle virus (MCMV) and a potyvirus, most often sugarcane mosaic virus (SCMV). This disease seriously threatens food security across sub-Saharan Africa (SSA). We investigated a major-effect quantitative trait locus for resistance on chromosome 6, named the maize lethal necrosis susceptibility locus 1 (qMLNS1), derived from the Thai line KS23-6. Fine mapping and CRISPR-Cas9 editing of the candidate genes within the narrowed 105 kb interval revealed a peroxisomal peptidase as the underlying cause of susceptibility. Confocal microscopy confirmed the localization of the MLNS1 protein within peroxisomes. Targeted knockout of the Mlns1 gene in the susceptible elite line CML536 from SSA conferred resistance comparable to KS23-6 in field trials conducted in Naivasha, Kenya. This knockout specifically blocked MCMV accumulation without affecting SCMV. The edited lines showed no yield penalty or agronomic defects under disease-free conditions. Our findings uncover a mechanistic link between a peroxisomal enzyme and viral susceptibility. They also establish a rapid, scalable gene editing strategy for incorporating MLN resistance into elite germplasm, offering a model for combating similar viral diseases in staple crops globally.},
}

*Plant Diseases/virology/genetics
*Zea mays/virology/genetics
*Disease Resistance/genetics
*Peroxisomes/enzymology/genetics
Potyvirus/pathogenicity
Gene Knockout Techniques
*Peptide Hydrolases/genetics/metabolism
Quantitative Trait Loci/genetics
Tombusviridae/pathogenicity
CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism

@article {pmid41927370,
year = {2026},
author = {Zheng, J and Wang, X and Wu, M and Liu, J and Feng, H and Yang, H and Li, D and Wang, H and Hu, J and Zuo, E},
title = {Large-scale parallel characterization of RNA-guided nuclease activity and specificity.},
journal = {Science bulletin},
volume = {71},
number = {8},
pages = {2044-2054},
doi = {10.1016/j.scib.2026.03.047},
pmid = {41927370},
issn = {2095-9281},
mesh = {*Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *Endonucleases/metabolism/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {As systematic comparisons of editing efficiency and specificity seldom keep pace with rapid developments in RNA-guided nucleases (RGNs), the current study examined 50 such editing systems and characterized the off-target effects and genomic structural impacts of a subset of high-efficiency RGNs. Among them, AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus demonstrated similar or higher efficiency compared to SpCas9, while the relatively high efficiency and small size of enOsCas12f1 together support its suitability for in vivo delivery. AsCpf1-YH and FnCpf1 exhibited the lowest single-guide RNA-dependent (sgRNA-dependent) off-target risks, whereas DpFNuc showed the highest. Genomic structural analysis revealed that enCas12f-HKRA frequently introduces chromosomal translocations, while Cas12j-SF05 poses a lower risk of such mutations. Notably, the high-efficiency RGNs were associated with translocation hotspots. Additionally, enRhCas12f1 and SpaCas12f1 had the lowest cytotoxicity, while enAsCpf1-HF strongly inhibited cell proliferation. This study establishes the first multidimensional performance evaluation framework for RGNs, providing a data-driven tool to support precise genome editing.},
}

*Gene Editing/methods
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems
*Endonucleases/metabolism/genetics
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42051075,
year = {2026},
author = {Vats, S and Jadhav, H and Mahakalkar, B and Patil, G and Sonah, H and Sharma, TR and Deshmukh, R},
title = {Genome Editing of a Carotenogenic Gene for Lycopene Enhancement Increases Heavy Metal Stress Susceptibility in Tomato (Solanum lycopersicum L.).},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70884},
doi = {10.1111/ppl.70884},
pmid = {42051075},
issn = {1399-3054},
support = {BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; //Anusandhan National Research Foundation, JC Bose Fellowship, Government of India./ ; HSCSIT/R&D/2024/511//Haryana State Council for Science Innovation and Technology (HSCSIT)/ ; },
mesh = {*Solanum lycopersicum/genetics/drug effects/physiology/metabolism ; *Gene Editing/methods ; *Lycopene/metabolism ; Stress, Physiological/genetics/drug effects ; *Metals, Heavy/toxicity ; Fruit/genetics ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Intramolecular Lyases/genetics/metabolism ; Gene Expression Regulation, Plant ; Carotenoids/metabolism ; Plants, Genetically Modified ; },
abstract = {Improving the nutritional quality and abiotic stress tolerance of crop plants is essential for sustainable agriculture and global food security. Recent advances in genome editing, particularly the CRISPR/Cas9 system, have enabled precise modification of metabolic pathways to enhance valuable traits such as carotenoid accumulation. The present study aimed to enhance fruit lycopene content and assess associated stress responses in tomato (Solanum lycopersicum L.) through targeted genome editing of the lycopene beta cyclase (β-LCY, EC 5.5.1.19) gene, encoding for a key enzyme in the carotenoid biosynthetic pathway. A Csy4-based multiplex CRISPR/Cas9 approach was applied to edit β-LCY in three tomato genotypes, including two cultivated varieties and the wild species S. peruvianum L. Genotypic analysis revealed significant genotype-dependent differences in editing efficiency. The β-LCY knockout lines exhibited markedly increased lycopene accumulation in fruits, resulting in enhanced pigmentation. However, when subjected to cadmium stress, these lines showed greater susceptibility than wild-type plants, with pronounced wilting and stress symptoms. Physiological, biochemical, and metabolomic analyses confirmed disruption of stress-response mechanisms associated with carotenoid pathway modification. These findings demonstrate that while genome editing can successfully enhance desirable metabolic traits, it may also impair abiotic stress tolerance. This study provides new insight into the complex interplay between the carotenoid biosynthetic pathway and stress adaptation in tomato.},
}

*Solanum lycopersicum/genetics/drug effects/physiology/metabolism
*Gene Editing/methods
*Lycopene/metabolism
Stress, Physiological/genetics/drug effects
*Metals, Heavy/toxicity
Fruit/genetics
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Intramolecular Lyases/genetics/metabolism
Gene Expression Regulation, Plant
Carotenoids/metabolism
Plants, Genetically Modified

@article {pmid42053263,
year = {2026},
author = {Turowski, P and Gatermann, SG and Pfennigwerth, N},
title = {Cefiderocol resistance mediated by mutation of the miniconductance mechanosensitive channel MscM in Klebsiella oxytoca.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {5},
pages = {},
doi = {10.1093/jac/dkag147},
pmid = {42053263},
issn = {1460-2091},
support = {//Robert Koch Institute/ ; 1369-402//German Ministry of Health/ ; },
mesh = {*Klebsiella oxytoca/drug effects/genetics/growth & development/isolation & purification ; *Anti-Bacterial Agents/pharmacology ; *Cephalosporins/pharmacology ; Microbial Sensitivity Tests ; Humans ; Klebsiella Infections/microbiology ; Cefiderocol/pharmacology ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Ion Channels/genetics ; *Bacterial Proteins/genetics ; Whole Genome Sequencing ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: To investigate the emergence of cefiderocol resistance in a clinical Klebsiella oxytoca isolate and to identify the underlying mechanism.

METHODS: A clinical isolate of K. oxytoca susceptible to cefiderocol was exposed to stepwise increasing cefiderocol concentrations via broth microdilution in iron-depleted CAMHB (ID-CAMHB) to select spontaneous mutants. WGS identified potential resistance-associated mutations. CRISPR-Cas9 genome editing was used to confirm causality. Growth curves in CAMHB and ID-CAMHB were performed to assess potential growth alterations.

RESULTS: A spontaneous mutant with elevated cefiderocol MIC (16 mg/L) carried a Q1008L substitution in the miniconductance mechanosensitive channel MscM. CRISPR-edited strains reproduced this phenotype. Growth kinetics did not reveal an obvious growth defect under the tested in vitro conditions.

CONCLUSION: This is the first report linking cefiderocol resistance to a mutation in MscM in K. oxytoca. Although observed in a single isolate, the lack of an apparent growth defect under the tested conditions suggests that this resistance mechanism may persist in the absence of antibiotic pressure.},
}

*Klebsiella oxytoca/drug effects/genetics/growth & development/isolation & purification
*Anti-Bacterial Agents/pharmacology
*Cephalosporins/pharmacology
Microbial Sensitivity Tests
Humans
Klebsiella Infections/microbiology
Cefiderocol/pharmacology
*Mutation
*Drug Resistance, Bacterial/genetics
*Ion Channels/genetics
*Bacterial Proteins/genetics
Whole Genome Sequencing
Gene Editing
CRISPR-Cas Systems

@article {pmid42053665,
year = {2026},
author = {Lei, M and Bakhsh, MZM and Zhang, X and He, D and Dai, C and Ma, C and Tu, J and Shen, J and Wen, J and Fu, T and Yi, B},
title = {From haploid inducer to CMS donor: repurposing of CENH3 to create a CMS line in a single step in Brassica napus.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {42053665},
issn = {1432-203X},
support = {2024ZD04077//Biological Breeding-National Science and Technology/ ; },
mesh = {*Haploidy ; *Brassica napus/genetics ; *Plant Infertility/genetics ; *Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; Cytoplasm/genetics ; CRISPR-Cas Systems/genetics ; *Histones/genetics/metabolism ; Mutation ; Centromere/genetics/metabolism ; },
abstract = {Mutation in centromere histone H3 (CENH3) protein could induce a paternal haploid with maternal cytoplasm in rapeseed. By paternal haploid induction, a cytoplasmic male sterile line can be created in any genetic background within one breeding cycle. Hybrid development in rapeseed relies primarily on the three-line system, which includes a cytoplasmic male sterile (CMS) line. Conventionally, these CMS lines are developed through backcrossing, a process that requires several breeding cycles to complete. More recently, the doubled haploid technique has been employed in various crops to generate homozygous lines within a single breeding cycle. In the present study, we utilized a haploid induction (HI) strategy to produce fertile homozygous lines and CMS lines via paternal haploid induction. We have created single homozygous and double heterozygous mutants of the BnaCENH3 gene in the rapeseed cultivar ganA (hau-CMS) using CRISPR/Cas9 technique. Upon hybridization of CMS-HI line with wild type can successfully induced paternal haploids with maternal sterile cytoplasm. This system offers the ability to introduce sterile cytoplasm into any genetic background within a single generation.},
}

*Haploidy
*Brassica napus/genetics
*Plant Infertility/genetics
*Plant Proteins/genetics/metabolism
Plant Breeding/methods
Cytoplasm/genetics
CRISPR-Cas Systems/genetics
*Histones/genetics/metabolism
Mutation
Centromere/genetics/metabolism

@article {pmid42053928,
year = {2026},
author = {Deres, D and Terefe, M},
title = {Beyond CRISPR/Cas9: emerging genome editing technologies for next-generation crop improvement.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42053928},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing has changed plant biology and accelerated crop improvement. CRISPR/Cas9 allows precise and efficient genetic changes in many species. Still, Cas9 has limits like PAM restrictions, off-target effects, and varying editing success. This led to new systems. Editors like Cas12a, CasΦ, CasMINI, and CasX offer more targeting options, can edit RNA, and work better with hard to edit plant genomes. Precision tools such as base editors and prime editors make precise changes by swapping nucleotides or adding small pieces without cutting both DNA strands. This improves accuracy. Beyond single tools, combined and step by step editing methods can be used for handling complex traits controlled by many genes. Using several methods like CRISPR knockouts, base and prime editing, epigenome editing and recombinase systems-breeders can improve traits while reducing unwanted side effects. Stepwise editing helps to test changes, confirm their effects, and improve entire biological pathways. Combining these approaches with AI-driven analysis, target prediction, and design optimization makes it easier to pick the best genes and edits for desired traits. These advanced editing methods are used to boost stress tolerance, fight diseases, improve nutrition, increase yields, and enhance quality after harvest. Despite progress, problems remain with how efficient edits can be made, delivering tools into plants, reliance on specific genotypes, unclear regulations, and acceptance by society. Looking ahead, joying genome editing with AI, fast breeding techniques help develop stronger, high yielding crops and support global food security.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics
*Genome, Plant/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics

@article {pmid42056528,
year = {2026},
author = {Marino, ND and Talaie, A and Gerovac, M and Rodriguez, JL and Schmidt, AD and Astmann, TJ and Carion, H and Taylor, AF and Liliedahl, J and Haniyur, S and Zoga, K and Johnson, MC and Buhlmann, L and Chen, KH and Silas, S and Yuping, L and Zhang, Y and Swaney, DL and Vogel, J and Bondy-Denomy, J},
title = {Translation-dependent degradation of cas12 mRNA triggered by an anti-CRISPR.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {42056528},
issn = {1476-4687},
abstract = {Bacteria encode diverse defence systems, including CRISPR-Cas, to recognize and cleave the DNA of bacteriophages (phages) and other mobile genetic elements[1]. In response, phages encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas activity by blocking DNA binding or cleavage[2]. Here we report an unexpected mechanism by which the anti-CRISPR AcrVA2 inhibits Cas12a biogenesis. AcrVA2 binds conserved and functionally important amino acid residues near the Cas12a N-terminus and triggers selective degradation of cas12a mRNA as it is translated. Additionally, conserved residues in the AcrVA2 C-terminal domain enable co-sedimentation with ribosomes and polysomes, which is required to achieve targeted co-translational mRNA degradation. The AcrVA2 C-terminal domain is broadly conserved in homologs encoded by diverse mobile genetic elements, typically in hosts that lack cas12a, suggesting that these homologues may recognize and downregulate alternative substrates in other bacteria. These findings reveal a novel mechanism for molecular conflict and gene regulation in bacteria.},
}

@article {pmid41839994,
year = {2026},
author = {Fu, X and Zhao, F and Ge, J and Guan, H and Lv, P and Guo, F and Ma, H and Ainiwaer, M and Hu, S},
title = {Establishment of a rapid Brucella detection method based on MCDA-CRISPR dual signal amplification system for reducing transfusion-transmitted diseases.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839994},
issn = {2045-2322},
support = {CXCY2025024//the Xinjiang Medical University Graduate Innovation and Entrepreneurship Project/ ; KYQDJJ2024006//the Doctoral Scientific Research Startup Fund of Zhengzhou Central Hospital/ ; 2022-2-6042//the Capital's Funds for Health Improvement and Research/ ; 2025YLZDJH193//The Guidance Plan Project for Scientific and Technological Innovation in the Medical and Health Field of Zhengzhou City/ ; },
mesh = {*Brucella/genetics/isolation & purification ; Humans ; *Brucellosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Blood Donors ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; China ; Blood Transfusion ; },
abstract = {Brucellosis is a common zoonotic disease caused by Brucella and remains a globally concerning public health issue. Timely and effective detection methods are crucial for clinical diagnosis. We developed a novel Brucella detection platform (MCDA-CRISPR) by integrating multiple cross displacement amplification (MCDA) with a CRISPR-Cas12a-based biosensing system, and preliminarily applied it for the first time to screen for Brucella in voluntary blood donors from Xinjiang, China. This technology enables amplification under isothermal conditions at 64 °C using only a water bath, requires no specialized equipment, and completes detection within 60 min. Amplification products can be directly visualized under UV light without complex interpretation. Performance results demonstrated a minimum detection limit of 1 fg/μL for Brucella DNA, making the method 100 times more sensitive than conventional PCR. The assay showed 100% specificity for Brucella detection with no cross-reactivity to non-Brucella pathogens. The assay could also detect Brucella in blood donors samples and showed the same sensitivity and specificity as the culture method. The assay is a visual, sensitive, and highly specific detection technique. When applied to routine blood transfusion screening in areas with high prevalence of brucellosis, such as Xinjiang, can effectively reduce the risk of transfusion-transmitted brucellosis, and hold broad application prospects in resource-limited primary or field testing scenarios.},
}

*Brucella/genetics/isolation & purification
Humans
*Brucellosis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems/genetics
Blood Donors
Sensitivity and Specificity
DNA, Bacterial/genetics
China
Blood Transfusion

@article {pmid41885428,
year = {2026},
author = {Carrington, E and Ballmer, D and Niederwieser, I and Thommen, BT and Brancucci, NMB and Voss, TS},
title = {A heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum.},
journal = {mSphere},
volume = {11},
number = {4},
pages = {e0088425},
doi = {10.1128/msphere.00884-25},
pmid = {41885428},
issn = {2379-5042},
support = {310030_184785,310030_220001//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; 310030_200683//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; ALTF 920-2024//European Molecular Biology Organization/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Drug Resistance/genetics ; Pyrimethamine/pharmacology ; Tetrahydrofolate Dehydrogenase/genetics ; Protozoan Proteins/genetics ; Antimalarials/pharmacology ; Thymidylate Synthase/genetics ; Humans ; Malaria, Falciparum/parasitology ; Plasmids ; Multienzyme Complexes ; },
abstract = {CRISPR/Cas9-based gene editing of the malaria parasite Plasmodium falciparum has emerged as a transformative tool for advancing functional studies on parasite biology and identifying new therapeutic targets. Currently applied CRISPR/Cas9 methodologies depend on a limited set of heterologous drug resistance markers for the selection of transgenic parasites, which restricts the potential for iterative genetic modifications. Here, we developed a heterologous marker-free CRISPR/Cas9 gene editing strategy (CRISPR/Cas9[pyrR]) for P. falciparum based on the simultaneous editing of a gene of interest and introduction of pyrimethamine (PYR) resistance-conferring mutations into the dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) gene. By providing a pfdhfr[pyrR] donor sequence and the Cas9 expression cassette on separate plasmids, CRISPR/Cas9[pyrR] ensures that only parasites acquiring both plasmids survive under PYR pressure. As a proof of principle, we applied CRISPR/Cas9[pyrR] to generate two transgenic parasite lines expressing GFP-tagged versions of the putative nuclear envelope protein PfGEX1 and nuclear pore protein PfNUP116, respectively. We show that PfGEX1-GFP marks the nuclear envelope specifically in gametocytes, but not in asexual blood stage parasites. Similarly, and against previous reports, we find PfNUP116-GFP expression is undetectable in asexual parasites but instead localizes to a distinct perinuclear region in early gametocytes. These results suggest dynamic compositional changes of the nuclear periphery during sexual differentiation. We further demonstrate sequential genetic engineering of the PfNUP116-GFP-expressing line using the human dhfr drug resistance marker combined with WR99210-based selection by additionally tagging PfAP2-G, the master transcriptional regulator of sexual commitment, and the nuclear pore protein PfNUP313. Hence, CRISPR/Cas9[pyrR] provides a versatile and effective new method that enhances and complements the current genetic toolkit for malaria research.IMPORTANCEMalaria tropica, which is caused by the unicellular parasite Plasmodium falciparum, is one of the most devastating infectious diseases worldwide. The development of urgently needed effective vaccines and new antimalarial drugs with novel modes of action requires a profound understanding of parasite biology. CRISPR/Cas9-based genome engineering is beyond doubt the most important experimental approach to study the function and essentiality of parasite proteins and to identify and validate new vaccine and drug targets. In this study, we developed and successfully applied a modified CRISPR/Cas9 strategy, termed CRISPR/Cas9[pyrR], that avoids the use of a heterologous drug resistance marker for the selection of genetically modified parasites. CRISPR/Cas9[pyrR] thus complements the CRISPR/Cas9 toolbox available for gene editing in P. falciparum and overcomes some of the limitations of currently employed protocols.},
}

*Plasmodium falciparum/genetics/drug effects
*CRISPR-Cas Systems
*Gene Editing/methods
Drug Resistance/genetics
Pyrimethamine/pharmacology
Tetrahydrofolate Dehydrogenase/genetics
Protozoan Proteins/genetics
Antimalarials/pharmacology
Thymidylate Synthase/genetics
Humans
Malaria, Falciparum/parasitology
Plasmids
Multienzyme Complexes

@article {pmid41964558,
year = {2026},
author = {Guo, A and Guo, W and Guo, Y and Zhang, Y and Zhang, Z and Zou, X and Sun, Z},
title = {Clustered Regularly Interspaced Short Palindromic Repeat-Based Colorimetric Aptasensor Combined with Smartphone Imaging and Deep Learning Enables Selective Recycling and Visual Prediction of Microplastics in the Environment.},
journal = {Analytical chemistry},
volume = {98},
number = {16},
pages = {11886-11898},
doi = {10.1021/acs.analchem.5c08138},
pmid = {41964558},
issn = {1520-6882},
mesh = {*Colorimetry/methods ; *Smartphone ; *Aptamers, Nucleotide/chemistry/genetics ; *Microplastics/analysis ; *Deep Learning ; Polystyrenes/analysis ; *Biosensing Techniques/methods ; Polyvinyl Chloride/analysis ; *Water Pollutants, Chemical/analysis ; CRISPR-Cas Systems ; },
abstract = {Microplastics present significant risks to human health and ecosystem stability, creating an urgent need for analytical methods that are simple, rapid, sensitive, and field-deployable. Herein, we report a clustered regularly interspaced short palindromic repeat (CRISPR)-based colorimetric aptasensor for the detection of poly(vinyl chloride) (PVC) and polystyrene (PS) microplastics. This platform leverages the high specificity of PVC and PS aptamers integrated into a Fe3O4@Au-DNA magnetic complex, which facilitates capture, separation, and detection. Upon microplastic binding, a competitive reaction releases an activator DNA, initiating a dual CRISPR-Cas12a system for signal amplification. The activated Cas12a trans-cleavage activity is then linked to a hemin-aptamer DNAzyme colorimetric reaction, converting the signal into a visible color change. This colorimetric output is captured by smartphone imaging and processed in real time. Furthermore, a deep-learning-based regression model was developed to enable the quantitative prediction of PVC and PS micro/nanoplastics in diverse environmental matrices. The method exhibited high selectivity and a broad dynamic range from 10[-2] to 10[3] μg/mL. In smartphone detection mode, the limits of detection for PVC and PS reached 3.1 ng/mL and 3.7 ng/mL, respectively. This approach significantly enhances detection performance and stability, enabling visual monitoring of microplastics in complex real samples. Collectively, this work provides a rapid and effective strategy for the extraction and real-time quantification of small molecules.},
}

*Colorimetry/methods
*Smartphone
*Aptamers, Nucleotide/chemistry/genetics
*Microplastics/analysis
*Deep Learning
Polystyrenes/analysis
*Biosensing Techniques/methods
Polyvinyl Chloride/analysis
*Water Pollutants, Chemical/analysis
CRISPR-Cas Systems

@article {pmid42046057,
year = {2026},
author = {Hsu, CY and Abdelgawwad El-Sehrawy, AAM and Alshkarchy, SS and Abdul, AS and Ganesan, S and Gupta, PK and Sharma, R and Nayak, PP and Ebrahimpour, A and Khazaei, Y},
title = {Innovative approaches in the treatment of hematologic malignancies: the role of CRISPR-engineered microbiomes along the gut-immune axis in immunotherapy development.},
journal = {Cancer cell international},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12935-026-04316-0},
pmid = {42046057},
issn = {1475-2867},
}

@article {pmid42048304,
year = {2026},
author = {Mallon, J and Lenihan, CJ and Shridhar, S and Bailey, S},
title = {Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system.},
journal = {The Biochemical journal},
volume = {},
number = {},
pages = {},
doi = {10.1042/BCJ20260189},
pmid = {42048304},
issn = {1470-8728},
abstract = {Type I-B CRISPR-Cas systems represent the most abundant CRISPR subtype in nature and have emerged as powerful tools for endogenous genome editing in diverse prokaryotes. Here we reconstitute and characterize the type I-B1 system from the thermophile Thermotoga maritima (Tma) using purified components. We demonstrate that Tma Cascade requires standalone Cas11 expression, as the cryptic internal translation start site within cas8b1 is non-functional in E. coli. The reconstituted system exhibits canonical type I function including RNA-guided DNA binding, PAM-dependent target discrimination, Cas3-mediated degradation, and seed region interrogation spanning seven PAM-proximal nucleotides. Using next-generation sequencing-based PAM library screens, we define a YYD consensus PAM (Y = C/T; D = G/A/T) with strong discrimination against the array repeat-adjacent sequence (AAC). Comprehensive PAM profiling reveals context-dependent tolerance for non-consensus sequences and identifies numerous intermediate-activity PAMs that may function in priming. Comparison with other characterized type I-B systems reveals correlation between Cas8b variant and position -3 specificity, conserved pyrimidine preference at position -2, and variability at position -1. This work establishes a thermostable type I-B platform for biotechnological applications and provides insights into evolutionary mechanisms balancing PAM promiscuity with self-discrimination in the most abundant CRISPR-Cas subtype.},
}

@article {pmid42048640,
year = {2026},
author = {Dominy, C},
title = {CRISPR Diagnostics, in Your Pocket.},
journal = {Journal of medical Internet research},
volume = {28},
number = {},
pages = {e98572},
doi = {10.2196/98572},
pmid = {42048640},
issn = {1438-8871},
}

@article {pmid42048928,
year = {2026},
author = {Li, L and Ju, R and Yan, Y and Lou, Y and Chen, H and Wen, Q and Wang, M and Lei, R and Liu, J and Wang, X},
title = {Nanomaterial-enabled CRISPR-Cas biosensing for non-nucleic acid targets: Strategies, mechanisms, and applications.},
journal = {Talanta},
volume = {307},
number = {},
pages = {129831},
doi = {10.1016/j.talanta.2026.129831},
pmid = {42048928},
issn = {1873-3573},
abstract = {Nanomaterial-assisted CRISPR-Cas biosensing has transcended nucleic acid detection, emerging as a prominent paradigm for the ultrasensitive analysis of non-nucleic acid targets. This review systematically elucidates recent advances by proposing a novel three-dimensional framework encompassing transduction efficiency, signal output, and application adaptation. We first focus on molecular engineering approaches that convert non-nucleic acid recognition events into CRISPR activation signals, critically assessing the inherent efficiency limits and bottlenecks of key techniques such as aptamer switches, nuclease allosteric effects, substituent steric hindrance, Cas protein allosteric regulation, and Cas protein engineering. We then detail how various nanomaterials, including AuNPs, QDs, UCNPs, MOFs, nanozymes, synergistically enhance the efficiency of optical, electrochemical, and multimodal signal output. Utilizing this framework, the review analyzes representative applications in detecting proteins, small molecules, and heavy metal ions, clarifying the mechanisms behind performance enhancement and current limitations. Finally, we discuss persistent challenges and prospectively proposes disruptive future directions, such as constructing CRISPR sensing networks, developing intelligent nanocarriers, and creating closed-loop theranostic systems. This study provides a technical overview, a critical framework, and a design roadmap, facilitating the rational design of biosensors and enabling the realization of integrated bio-regulatory platforms.},
}

@article {pmid42049209,
year = {2026},
author = {Da Lage, JL and Bonneau, M and Moreno, C and Le Rouzic, A},
title = {Is the alpha-amylase paralogue Amyrel dispensable in Drosophila melanogaster?.},
journal = {Open biology},
volume = {16},
number = {4},
pages = {},
doi = {10.1098/rsob.250411},
pmid = {42049209},
issn = {2046-2441},
support = {//CNRS/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism/enzymology ; *Drosophila Proteins/genetics/metabolism ; Female ; *alpha-Amylases/genetics/metabolism ; Male ; CRISPR-Cas Systems ; },
abstract = {Divergent duplicated gene copies are considered to get new or variant function or regulation through sub- or neofunctionalization. In Drosophila and other flies (Muscomorpha), the alpha-amylase paralogue Amyrel is known to have peculiar enzymological properties compared with the classical enzyme Amy. Yet, its real function in fly biology is unclear. Here, we show that Amyrel and Amy share similar regulation patterns such as sugar downregulation and midgut-specific expression in Drosophila melanogaster. Most regulatory information lies within 500 bp of the upstream sequence, as enhanced green fluorescent protein expression under the Amyrel promoter mimics Amyrel expression quite well. To get an insight into Amyrel function, we knocked out the gene using CRISPR-Cas9. Setting a competition experiment between wild-type (wt) and null alleles over 40 generations, we estimated the selective advantage of the wt to be 2%. However, Amyrel-null mutant lines exhibited no clear defect in several life history traits. Interestingly, while Amyrel had very low expression in young adults, it was significantly upregulated in females aged two months; however, lifespan was not affected. Overall, we were able to document substantial functional and regulatory differences between the Amyrel copy and the regular amylase, and we showed that carrying an Amyrel gene conferred a competitive fitness advantage.},
}

Animals
*Drosophila melanogaster/genetics/metabolism/enzymology
*Drosophila Proteins/genetics/metabolism
Female
*alpha-Amylases/genetics/metabolism
Male
CRISPR-Cas Systems

@article {pmid42049231,
year = {2026},
author = {Zhou, J and Wang, X and Zhou, L and Zhang, H and Ye, S and Yuan, YJ},
title = {Automated linear DNA assembly of A. thaliana's chloroplast and mitochondrial genome.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag380},
pmid = {42049231},
issn = {1362-4962},
support = {2021YFA0909300//National Key Research and Development Program of China/ ; //Ministry of Science and Technology of the People's Republic of China/ ; 22527901//National Natural Science Foundation of China/ ; //National Major Research Instrumentation Program/ ; JYB2025XDXM503//Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China/ ; 25ZXWCSY00330//Tianjin Municipal Science and Technology Major Program/ ; },
mesh = {*Arabidopsis/genetics ; *Genome, Mitochondrial ; *Genome, Chloroplast ; Escherichia coli/genetics ; Genome, Plant ; Synthetic Biology/methods ; CRISPR-Cas Systems ; },
abstract = {Synthetic genomics is advancing from microbial toward multicellular organisms. However, current manual methods for DNA and genome assembly remain inadequate for the efficient, large-scale production of long DNA constructs. Here, we present Programmed DNA Assembly via Cas9 and Conjugative Transfer (PACT), a method that integrates a linear vector system, bacterial conjugation, and programmable Cas9-mediated cleavage to achieve highly efficient, iterative assembly of large DNA fragments. PACT enhances assembly efficiency by ~30-fold compared to conventional circular vector strategies, enabling one-step assembly of DNA up to 80 kb. We engineered four single guide-RNA-Marker donor cassettes to support iterative assembly workflows. PACT can utilize low-recombination Escherichia coli strains as hosts to efficiently assemble Arabidopsis thaliana mitochondrial genome with high repeat units. Integrated with an automated robotic platform, we developed an unattended, high-throughput pipeline (aPACT) toward large-scale parallel DNA assembly. Using aPACT, we successfully assembled three large DNA constructs: a 210 kb digital DNA, the designed chloroplast (120 kb) and mitochondrial (350 kb) genome of A. thaliana. This automated system offers a powerful tool for scalable assembly of large DNA molecules, accelerating synthetic genomics research toward complex multicellular organisms.},
}

*Arabidopsis/genetics
*Genome, Mitochondrial
*Genome, Chloroplast
Escherichia coli/genetics
Genome, Plant
Synthetic Biology/methods
CRISPR-Cas Systems

@article {pmid41820368,
year = {2026},
author = {Bertlin, JAC and Pauzaite, T and Liang, Q and Wit, N and Williamson, JC and Sia, JJ and Matheson, NJ and Ortmann, BM and Mitchell, TJ and Speak, AO and Zhang, Q and Nathan, JA},
title = {VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41820368},
issn = {2041-1723},
support = {R01 CA284591/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Membrane Proteins/metabolism/genetics ; Cell Line, Tumor ; Animals ; Signal Transduction ; Mice ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Interferon Type I/metabolism ; *Synthetic Lethal Mutations ; STING Protein ; },
abstract = {Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.},
}

Humans
*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism
*Carcinoma, Renal Cell/genetics/metabolism/pathology
*Kidney Neoplasms/genetics/metabolism/pathology
*Membrane Proteins/metabolism/genetics
Cell Line, Tumor
Animals
Signal Transduction
Mice
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Interferon Type I/metabolism
*Synthetic Lethal Mutations
STING Protein

@article {pmid41955813,
year = {2026},
author = {Ji, Z and Zhao, Y and Huang, J and Shan, Y and Li, J and Huang, Q and Wu, M and Liu, F},
title = {Portable amplification-free digital droplet CRISPR/Cas12a platform for one-pot multiplexed viruses detection with attomolar sensitivity.},
journal = {Biosensors & bioelectronics},
volume = {305},
number = {},
pages = {118668},
doi = {10.1016/j.bios.2026.118668},
pmid = {41955813},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; Animals ; Swine ; *Circovirus/isolation & purification/genetics ; Limit of Detection ; *Porcine epidemic diarrhea virus/isolation & purification/genetics ; *Porcine respiratory and reproductive syndrome virus/isolation & purification/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {High-throughput, sensitive, and field-deployable nucleic acid detection is essential for timely pathogen surveillance. Here, we developed a novel amplification-free and portable platform based on an instrument-free polydisperse digital droplet CRISPR/Cas12a (DD-Cas12a) assay, enabling the simultaneous detection of three synthetic nucleic acid targets in a single reaction. The platform achieved attomolar-level sensitivity (0.15 fM) without pre-amplification, generated detection results within 40 min, and maintained high specificity even in the presence of a 10-fold excess of non-target nucleic acids. The DD-Cas12a was further applied to detect one DNA virus, porcine circovirus type 2 (PCV2), and two RNA viruses, porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection limits for PCV2, PEDV, and PRRSV were 126.0, 673.5, and 584.5 fg/μL, respectively, corresponding to sensitivity comparable to qPCR for PCV2 and 10-fold higher sensitivity for PEDV and PRRSV. To improve analytical throughput and objectivity, we established an Algorithm-Driven DD-Cas12a (ADC) platform for automated image analysis and result decoding. The custom-designed miniaturized portable instrument was developed for signal acquisition and field deployment. By combining the DD-Cas12a assay with the ADC platform and the portable instrument, we established a portable, digital, amplification-free, and ultrasensitive system for multiplex pathogen detection, providing a powerful tool for diagnostics, biosurveillance, and environmental monitoring.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/instrumentation
Animals
Swine
*Circovirus/isolation & purification/genetics
Limit of Detection
*Porcine epidemic diarrhea virus/isolation & purification/genetics
*Porcine respiratory and reproductive syndrome virus/isolation & purification/genetics
Nucleic Acid Amplification Techniques

@article {pmid41962435,
year = {2026},
author = {Jia, M and Ru, X and Bing, X and Zhai, F and Huang, M and Du, X},
title = {CRISPR/Cas12a-mediated aggregation of unmodified AuNPs via microwave-assisted heating-dry for label-free detection of Escherichia coli O157:H7.},
journal = {Biosensors & bioelectronics},
volume = {305},
number = {},
pages = {118676},
doi = {10.1016/j.bios.2026.118676},
pmid = {41962435},
issn = {1873-4235},
mesh = {*Escherichia coli O157/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Gold/chemistry ; Microwaves ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Colorimetry/methods ; Endodeoxyribonucleases/chemistry ; *Escherichia coli Infections/microbiology/diagnosis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for developing next-generation biosensors, yet achieving rapid, instrument-free visual detection remains a challenging. Conventional CRISPR-AuNP assays rely on pre-functionalized, thiol-modified gold nanoparticles (AuNPs), which introduce steric hindrance that slows Cas12a kinetics and increases cost and complexity. Here, we developed a rapid, label-free colorimetric platform that integrates a mismatched catalytic hairpin assembly (MCHA) for low-background amplification with CRISPR/Cas12a trans-cleavage, coupled to a microwave-assisted dry-heating method for in-situ probe conjugation. Unlike conventional approaches where CRISPR acts on pre-conjugated probes, our method allows Cas12a to cleave free, non-thiolated ssDNA probes. Intact probes rapidly conjugate to unmodified AuNPs under microwave irradiation via their high-affinity domain, forming a protective corona that prevents salt-induced aggregation. Conversely, probes that have been cleaved by target-activated CRISPR/Cas12a lose this conjugation ability, rendering AuNPs susceptible to aggregation and producing a visible color change. To address the conflicting ionic requirements between the CRISPR reaction and AuNP stability, a Ba(OH)2-mediated ionic reset step is introduced to remove excess Mg[2+] prior to probe conjugation. This platform enables the sensitive detection of Escherichia coli O157:H7 within 2 h a limit of detection of 5 CFU/mL and robust performance in complex food and clinical matrices. Combined with smartphone-based RGB analysis, the instrument-minimized strategy eliminates the need for chemical modification of AuNPs and specialized operators, offering a versatile, low-cost, and user-friendly platform well-suited for pathogen detection in resource-limited settings.},
}

*Escherichia coli O157/isolation & purification/genetics
*Biosensing Techniques/methods
*Gold/chemistry
Microwaves
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
Limit of Detection
Colorimetry/methods
Endodeoxyribonucleases/chemistry
*Escherichia coli Infections/microbiology/diagnosis
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41990423,
year = {2026},
author = {Wang, Q and Zhu, Y and Chen, B and Zhai, C and Xu, J and Xia, J},
title = {Asymmetric RPA-primed hybridization chain reaction enabling one-input-multiple-Cas12a activation for ultrasensitive Salmonella detection.},
journal = {Biosensors & bioelectronics},
volume = {305},
number = {},
pages = {118689},
doi = {10.1016/j.bios.2026.118689},
pmid = {41990423},
issn = {1873-4235},
mesh = {*Salmonella/isolation & purification/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Microbiology ; DNA, Single-Stranded/chemistry/genetics ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Bacterial Proteins/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; Endodeoxyribonucleases ; },
abstract = {Ensuring food safety requires accurate, simple detection of pathogens such as Salmonella. However, conventional methods suffer from long processing times, complicated procedures, and inadequate accuracy. To address this, we developed an integrated platform combining asymmetric recombinase polymerase amplification (aRPA), programmable hybridization chain reaction (HCR), and CRISPR/Cas12a readout in a simplified single-tube integrated format. Unlike conventional CRISPR assays, in which a single target activates a single Cas12a complex in a PAM-dependent manner, our design uses aRPA-generated single-stranded DNA to trigger HCR, converting each target into a long dsDNA molecule with multiple PAM sites. This architecture enables one-input, multiple-Cas12a activation, representing a shift from linear to cooperative amplification. Under optimized conditions, the assay achieves a detection limit of 1.8 × 10[2] colony-forming units/mL (CFU/mL) in direct screening, and reaches as low as 5 CFU/mL after a 6 h enrichment step. The method shows high specificity toward common foodborne pathogens and reliable performance in spiked food samples. Furthermore, a DNA releaser simplifies sample preparation and enhances convenience. In summary, this integrated strategy offers a reliable and practical tool for food safety monitoring.},
}

*Salmonella/isolation & purification/genetics
*Biosensing Techniques/methods
Nucleic Acid Hybridization
Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems/genetics
Limit of Detection
Food Microbiology
DNA, Single-Stranded/chemistry/genetics
DNA, Bacterial/genetics
Food Contamination/analysis
Bacterial Proteins/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
Endodeoxyribonucleases

@article {pmid41999797,
year = {2026},
author = {Wang, Z and Zhou, L and Ming, L},
title = {From "Simulation" to "Mirror": Gene editing and humanization redefines the next-generation precision oncology animal model.},
journal = {Gene},
volume = {998},
number = {},
pages = {150161},
doi = {10.1016/j.gene.2026.150161},
pmid = {41999797},
issn = {1879-0038},
mesh = {*Gene Editing/methods ; Animals ; Humans ; *Precision Medicine/methods ; Disease Models, Animal ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems ; Tumor Microenvironment/genetics ; Mice ; Xenograft Model Antitumor Assays/methods ; Mutation ; },
abstract = {Patient-derived xenograft (PDX) models, although conventionally used in oncology, exhibit critical limitations: they frequently lose patient-specific genetic mutations and lack the human leukocyte antigen (HLA) diversity essential for immune recognition, and fail to recapitulate the human tumor microenvironment (TME). These deficiencies contribute to immunotherapy prediction failure rates exceeding 80% in clinical translation. To address these gaps, we propose a Tumor Model 2.0 framework. This framework integrates multi-omics data (whole-genome, transcriptomic, and proteomic) with precision genome editing technologies (CRISPR-Cas9 and Prime Editing) to reconstruct patient-specific mutations across multiple biological layers. Employing an organoid-animal coupling platform with stepwise immune system construction and microenvironment remodeling-subsequently validated in large animals-the framework enables the creation of programmable, patient-specific digital twins. These high-fidelity models support personalized N-of-1 clinical trials, bridging the gap between preclinical research and clinical precision oncology.},
}

*Gene Editing/methods
Animals
Humans
*Precision Medicine/methods
Disease Models, Animal
*Neoplasms/genetics/therapy
CRISPR-Cas Systems
Tumor Microenvironment/genetics
Mice
Xenograft Model Antitumor Assays/methods
Mutation

@article {pmid42037564,
year = {2026},
author = {de Barros Rodrigues, DK and Leeuwerik, M and Brankovics, B and Fava, WS and Venturini, J and Meyer, W and Arai, T and Majima, H and Watanabe, A and de Souza Carvalho Melhem, M},
title = {Environmental Circulation of Aspergillus fumigatus With Reduced Susceptibility to Agricultural Triazole in Brazil: Clonal Dissemination of Potentially Resistant Genotypes.},
journal = {Mycoses},
volume = {69},
number = {5},
pages = {e70179},
doi = {10.1111/myc.70179},
pmid = {42037564},
issn = {1439-0507},
support = {201422/2024-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 443813/2023-0//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 88887.658397/2021-00//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; 88887.819536/2023-00//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; },
mesh = {*Aspergillus fumigatus/genetics/drug effects/isolation & purification/classification ; *Triazoles/pharmacology ; *Drug Resistance, Fungal/genetics ; Genotype ; *Antifungal Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Brazil/epidemiology ; Aspergillosis/microbiology ; Fungal Proteins/genetics ; Whole Genome Sequencing ; Microsatellite Repeats ; Agriculture ; Cytochrome P-450 Enzyme System/genetics ; Tubulin/genetics ; Polymorphism, Single Nucleotide ; Air Microbiology ; Mutation ; },
abstract = {BACKGROUND: Aspergillus fumigatus resistance to triazole antifungals poses an increasing global health concern. Moreover, the cross-resistance between azole antifungal agents used in clinical settings and those applied in agriculture has become an important emerging issue.

OBJECTIVES: In this study, we investigated the five environmental A. fumigatus strains showing reduced susceptibility to tebuconazole.

METHODS: Fungal strains were recovered from air samples collected around the homes of two patients with suspected aspergillosis caused by resistant isolates. Species identification was performed by sequencing the β-tubulin gene, and minimum inhibitory concentrations were determined by broth microdilution. The cyp51A gene was sequenced to detect mutations, and CRISPR-Cas9 genome editing was employed to investigate their influence on susceptibility patterns. Microsatellite genotyping was performed to assess genetic variability, followed by whole genome sequencing and single nucleotide polymorphism analysis.

RESULTS: The environmental strains presented the same cyp51A genotype characterised by the M172V substitution and silent mutations. Microsatellite genotyping and whole genome sequencing confirmed that the strains were clonal. Functional validation demonstrated that the M172V and silent mutations partially contribute to reduced susceptibility to tebuconazole but are not the main mechanism of resistance involved. Analysis of polymorphisms in genes other than cyp51A revealed no resistance-conferring mutations.

CONCLUSIONS: The findings described herein suggest the possibility of local clonal dissemination of environmental strains under selective pressure from agricultural azoles in a major agribusiness region of the Midwest of Brazil. This study highlights the silent spread of potentially resistant genotypes in urban areas and reinforces the need for environmental surveillance and expanded genomic monitoring in South America.},
}

*Aspergillus fumigatus/genetics/drug effects/isolation & purification/classification
*Triazoles/pharmacology
*Drug Resistance, Fungal/genetics
Genotype
*Antifungal Agents/pharmacology
Microbial Sensitivity Tests
Humans
Brazil/epidemiology
Aspergillosis/microbiology
Fungal Proteins/genetics
Whole Genome Sequencing
Microsatellite Repeats
Agriculture
Cytochrome P-450 Enzyme System/genetics
Tubulin/genetics
Polymorphism, Single Nucleotide
Air Microbiology
Mutation

@article {pmid42040301,
year = {2026},
author = {Iqbal, Z and Awan, AZ and Atta, S and Hussain, K and Khurshid, M and Ahmad, F and Munir, M and Ghafoor, A and Hashedi, SAA and Ramadan, KMA and El-Gananiny, SM and AlSaleh, MA},
title = {Unveiling the landscape of plant virology in Saudi Arabia: seven decades of progress and future directions toward Vision 2030.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1758142},
pmid = {42040301},
issn = {1664-462X},
abstract = {Plant viruses pose a persistent and escalating threat to global agriculture and food security, inflicting over $30 billion in annual losses - a challenge acutely felt in Saudi Arabia as it strives for agricultural self-sufficiency under Vision 2030. This is the first comprehensive review which presents seven decades of plant virology research in the Kingdom, from early symptom-based diagnosis to advanced molecular, genomic, and bioinformatics advances. A total of ~81 plant viral species infecting 46 plant host species have been documented across the major agroecological regions, dominated by positive-sense single-stranded RNA viruses (~70%). Among these viruses, some are economically most destructive-including alfalfa mosaic virus, cucumber mosaic virus, soilborne cereal mosaic virus, tomato yellow leaf curl virus, zucchini yellow mosaic virus, watermelon chlorotic stunt virus, and barley mild mosaic virus-posing recurrent challenges to key crops such as alfalfa, cucurbits, and tomatoes. Network analysis of virus distribution revealed strong epidemiological linkages among central and western agricultural regions, possibly driven by intensive cultivation and vector ecology. The review highlights emerging management strategies including CRISPR-Cas diagnostics, RNA interference, AI-based detection, nanotechnology, and plant growth promoting rhizobacteria. Gaps persist in genomic surveillance, vector ecology, and biosecurity enforcement. The review concludes with future research priorities emphasizing innovation, interdisciplinary collaboration, and the development of a national plant virus genomic and surveillance framework to secure sustainable agriculture in line with Vision 2030.},
}

@article {pmid42041587,
year = {2026},
author = {Elias, A and Stern, S},
title = {Gene Editing Strategies for Neurological and Mental Disorders: Advances in Delivery, Methodology, and Clinical Translation.},
journal = {Cells},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/cells15080720},
pmid = {42041587},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; *Nervous System Diseases/therapy/genetics ; *Mental Disorders/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Translational Research, Biomedical ; },
abstract = {Neurological and mental disorders are among the main causes of disability worldwide, affecting over three billion people and increasing the socioeconomic burden. Advances in molecular genetics and genome engineering have led to gene-targeted therapies that address root causes rather than just symptoms. This review covers current genome-editing tools, including CRISPR/Cas, base editing, and prime editing. The focus is on the benefits of gene editing in the central nervous system, where post-mitotic neurons allow lasting effects after a single treatment. It also discusses emerging delivery platforms such as viral vectors, nanoparticles, and exosome systems, as well as methods to bypass the blood-brain barrier. Recent clinical progress in spinal muscular atrophy, Parkinson's disease, Huntington's disease, and Alzheimer's disease is highlighted, with promising preclinical results for autism, bipolar disorder, epilepsy, and other neurogenetic conditions. The review concludes with regulatory issues, market trends, and ongoing clinical trials, underscoring the potential of gene therapies to transform disease management and provide long-term solutions.},
}

Humans
*Gene Editing/methods
*Nervous System Diseases/therapy/genetics
*Mental Disorders/therapy/genetics
*Genetic Therapy/methods
Animals
CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
*Translational Research, Biomedical

@article {pmid42042483,
year = {2026},
author = {Zhang, C and Wang, YX and Liu, XD and Iqbal, A and Wang, Q and Wang, Y},
title = {Integrated Pest Management Strategies for Controlling Phthorimaea (Tuta) absoluta: Advances in Biological, Pheromone, and Cultural Control Methods.},
journal = {Insects},
volume = {17},
number = {4},
pages = {},
doi = {10.3390/insects17040441},
pmid = {42042483},
issn = {2075-4450},
support = {No. XJ202411439006 and No. XJ202411439010.//Jilin Agricultural Science and Technology College Student Innovation and Entrepreneurship Training Program Project and the Jilin Agricultural Science and Technology University College Student Innovation and Entrepreneurship Training Program Project/ ; },
abstract = {The tomato leaf miner, Phthorimaea (Tuta) absoluta, Meyrick 1917 is recognized as a highly destructive pest, causing significant economic losses to crops in both greenhouse and open field environments across four continents: Asia, Africa, Europe, and South America. High genetic homogeneity among populations from various regions and countries indicates significant gene flow between P. absoluta populations, suggesting a lack of geographical barriers to dispersion. Furthermore, P. absoluta has developed resistance to insecticides due to target-site mutations or metabolic resistance, which enable the insect to withstand lethal doses of insecticides. To control this insect pest, the plant-mediated RNA interference (RNAi) is most promising host-induced gene silencing technique, utilized the plant's machinery to express double-stranded (dsRNA), which triggers the RNAi pathway in P. absoluta. Due to thermal tolerance, the P. absoluta has increased its area of invasion by 600 km per year over 9 years. Female P. absoluta releases pheromones that are recognized by males with a sophisticated olfactory circuit on their antenna. Pheromone binding proteins (PBPs) play a crucial role in mate recognition and attraction, and their expression peaks during courtship, specifically around 6:00 a.m. Given its potential to significantly alter the insect genome, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) offer a revolutionary strategy to control P. absoluta. Furthermore, this pest has developed remarkable adaptations to survive on unfavorable hosts by secreting specific proteins from its salivary glands that detoxify plant defenses. Insecticide resistance is likely the cause of field control failures of P. absoluta. Biological control, sex pheromone traps, and cultural control are the most promising approaches to address insecticide resistance resulting from these failures. Therefore, the implementation of integrated control programs and appropriate resistance management strategies is necessary to keep P. absoluta infestations under economic damage thresholds.},
}

@article {pmid42043550,
year = {2026},
author = {Yao, X and Yao, X and Luo, M and Luo, L and Zhou, L and Li, X},
title = {Establishment of a CRISPR-Cas12a based electrochemical detection method for Burkholderia gladioli and its subspecies cocovenenans in fresh noodles and tremella.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42043550},
issn = {1432-072X},
support = {2024CZ19//Science and Technology Plan Project of Administration for Market Regulation of Guangdong Province/ ; },
mesh = {*Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Burkholderia gladioli/isolation & purification/genetics/classification ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Burkholderia gladioli pv. cocovenenans is a pathogenic bacterium of concern due to its potential threat to food safety. This study presents a rapid electrochemical detection method based on dual recombinase-aided amplification (RAA) coupled with CRISPR-Cas12a for the sensitive and specific identification of B. gladioli and its toxigenic subspecies B. gladioli pv. cocovenenans in food. The 16 S rDNA and bonA genes were selected as species- and subspecies-specific targets, respectively, and corresponding CRISPR-Cas12a reaction systems were established. An electrochemical biosensor incorporating a gold electrode functionalized with single-stranded DNA probes was constructed, and its specificity and sensitivity were evaluated using artificially contaminated fresh noodles and tremella samples. The method precisely distinguished B. gladioli and its toxigenic cocovenenans subspecies within 1 h, achieving a limit of detection limit of 10[2] CFU/g in both food matrices. This strategy provides a rapid and field-deployable approach for distinguishing non-toxigenic and toxigenic B. gladioli strains in food, supporting timely screening and food safety monitoring.},
}

*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*Burkholderia gladioli/isolation & purification/genetics/classification
*CRISPR-Cas Systems
*Food Microbiology/methods
Sensitivity and Specificity
Bacterial Proteins/genetics
Nucleic Acid Amplification Techniques/methods
DNA, Bacterial/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42043584,
year = {2026},
author = {Ray, S and Vijayan, J and Vanchinathan, S and Dhakar, R and Nasrullah, N and Nagar, S and Dutta, TK and Chinnusamy, V},
title = {CRISP-PTG-Assembler Ver. 1.0: a primer design tool for polycistronic tRNA-gRNA (PTG) assembly for Cas9-based multiplex genome editing in plants.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42043584},
issn = {1432-2048},
support = {CRG/2021/002949//Science and Engineering Research Board (SERB), GoI/ ; Genome Editing Project//Indian Council of Agricultural Research/ ; Crop Science EFC-10//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *RNA, Transfer/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; DNA Primers/genetics ; },
abstract = {Multiplex genome editing using the CRISPR/Cas9 system allows simultaneous modifications at several genomic sites, offering great potential for crop improvement. Among various approaches, the polycistronic tRNA-gRNA (PTG) system is widely adopted due to its use of the host's native tRNA processing machinery, enabling the generation of multiple sgRNAs from a single transcript without the need for expressing any foreign RNA processing enzymes or ribozymes. However, designing the complete set of primers suitable for performing in vitro PTG assembly is complex and needs expertise, as a single mistake can lead to complete failure of the assembly process or subsequent editing. To overcome this challenge, we developed CRISP-PTG-Assembler Ver. 1.0, a user-friendly tool that takes only (i) 20-nucleotide sgRNA spacers and (ii) 4-nucleotide joiners as inputs; and produces colour-coded outputs in forms of (i) Primer-set required for complete PTG assembly, (ii) Primary PCR Amplicons, (iii) Overlap-Extension PCR Amplicons and (iv) Expected PTG assembly, for easy interpretation and construct making. Our novel assembly approach provides flexibility in sticky-end choice during golden gate ligation and ensures the fidelity of component sgRNAs in the PTG assembly by buffering against ligation errors (~ 1.5-40%) that may occur during the Golden Gate assembly process, thereby safeguarding the functionality of the in vivo-generated individual sgRNA molecules. We validated its effectiveness by editing two loci of the matrix metalloproteinase 1 gene in rice and demonstrated its applicability across various plant systems. With an intuitive interface and robust features, CRISP-PTG-Assembler empowers researchers of all levels to effectively implement PTG-based multiplex genome editing in plants.},
}

*Gene Editing/methods
*RNA, Transfer/genetics
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Plant/genetics
DNA Primers/genetics

@article {pmid42045369,
year = {2026},
author = {Ragucci, AE and Antine, SP and Leviss, EM and Mooney, SE and Garcia, JM and Shyrokova, L and Hauryliuk, V and Lee, ASY and Kranzusch, PJ},
title = {Nuclease-NTPase antiphage defence systems use conserved molecular features to control bacterial immunity.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42045369},
issn = {2058-5276},
support = {1DP2GM146250-01//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
abstract = {Bacteria encode diverse defence systems, including restriction-modification and CRISPR-Cas, that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate that many recently identified antiphage defence operons comprise a nuclease and NTPase protein, suggesting that additional nucleic acid-targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyse 16 nuclease-NTPase systems and define molecular features that control antiphage defence. Purification, biochemical characterization and in vitro reconstitution of nucleic acid degradation demonstrates that protein-protein complex formation is a shared feature of multigene nuclease-NTPase systems. We show that PaAbpAB, BtHachiman and EcPD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of antiphage defence system function and highlight the mechanistic diversity of nuclease-NTPase systems in bacterial immunity.},
}

@article {pmid41553078,
year = {2026},
author = {Xu, T and Wang, T and Zhang, C and Cao, Y and He, X},
title = {Bibliometric-Based Analysis of Global Trends and Collaborative Networks in Plant Genetic Engineering (1994-2024).},
journal = {Plant biotechnology journal},
volume = {24},
number = {5},
pages = {3065-3083},
doi = {10.1111/pbi.70550},
pmid = {41553078},
issn = {1467-7652},
support = {2023ZD0406310//Science and Technology Innovation 2030-Biological Breeding-Major Projects/ ; },
mesh = {*Bibliometrics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/trends ; Gene Editing ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; },
abstract = {Agricultural sustainability faces serious challenges from population growth, climate change and ecological degradation. Genetic modification (GM) technology can be regarded as a precise extension of the Green Revolution, aiming to balance yield enhancement with ecological integrity through biotechnology. To systematically examine global trend, this study conducts a bibliometric analysis using worldwide literature data from 1994 to 2024. The findings reveal a dual-core structure of international collaboration, centered on China and the United States. The United States is closely connected with Korea, Japan and the United Kingdom, forming a high-density cluster, while China engages with emerging regions in Southeast Asia and Africa through the Belt and Road Initiative. This initiative is intended to strengthen China's influence and is accompanied by the proliferation of technology in countries less endowed with resources. The technology lifecycle has been evolved through three distinct phases. Initially, the process of Agrobacterium-mediated transformation in tobacco plants was carried out, marking the beginning of transgenic development. This was followed by the implementation of RNA interference (RNAi) technology to silence multiple genes. Finally, a breakthrough happened through the development of CRISPR-Cas9 genome editing technologies. The analyses conducted in this study demonstrate the preponderance of CRISPR in contemporary research, thus suggesting that the industry places a premium on technological refinement. Hence, the future technological trajectory is predicted to focus on germplasm digitization, multi-gene editing, intelligent breeding and synthetic biology. Transgenic technology will serve as a foundational support for achieving sustainable food security in the forthcoming second green revolution.},
}

*Bibliometrics
Plants, Genetically Modified/genetics
*Genetic Engineering/trends
Gene Editing
CRISPR-Cas Systems
Crops, Agricultural/genetics

@article {pmid41825104,
year = {2026},
author = {Li, P and Wang, L and Li, G and Li, R and Li, Y and Zhang, Z and Yang, S and Tang, H and Liu, Z},
title = {Transcriptomic and phenotypic analysis of maize with CRISPR/Cas9-mediated targeted mutagenesis of melatonin synthesis genes under drought stress.},
journal = {Plant physiology and biochemistry : PPB},
volume = {233},
number = {},
pages = {111189},
doi = {10.1016/j.plaphy.2026.111189},
pmid = {41825104},
issn = {1873-2690},
mesh = {*Melatonin/biosynthesis/genetics ; *Zea mays/genetics/metabolism/physiology ; *Droughts ; *CRISPR-Cas Systems/genetics ; *Transcriptome ; *Stress, Physiological/genetics ; Abscisic Acid/metabolism ; Reactive Oxygen Species/metabolism ; Gene Expression Regulation, Plant ; Mutagenesis ; Plant Proteins/genetics/metabolism ; Phenotype ; Genes, Plant ; Gene Expression Profiling ; },
abstract = {Melatonin, a pleiotropic regulatory factor, plays a key role in mediating crop drought resistance. Herein, we conducted an integrated physiological and transcriptomic approach to elucidate the mitigating effect of endogenous melatonin in mitigating drought stress in maize. We generated a comt snat asmt maize mutant via CRISPR-Cas9-mediated simultaneous editing of ZmCOMT, ZmSNAT, and ZmASMT1-genes encoding rate-limiting enzymes in the endogenous melatonin biosynthesis pathway. Sequencing of the mutant lines revealed key amino acid substitutions (Gly168→Ala in ZmSNAT, Asp175→Glu in ZmCOMT, and Asp150→Glu in ZmASMT1) within critical protein domains, resulting from CRISPR-induced small insertions or deletions (indels), which led to subtle alterations in the tertiary conformation of corresponding proteins. These modifications resulted in an 86.70% increase in endogenous melatonin content. Under drought stress, the comt snat asmt maize exhibited enhanced antioxidant enzyme activities, leading to a significant reduction in reactive oxygen species (ROS) accumulation compared to the control. Furthermore, endogenous levels of melatonin, abscisic acid (ABA), cytokinin (CTK), and auxin (IAA) were markedly elevated, whereas gibberellin (GA) content was significantly reduced. Consistently, the activities of SNAT, ASMT, and COMT were also enhanced in the mutant. Transcriptomic profiling further revealed that endogenous melatonin regulates ABA, IAA, CTK, and GA signaling pathways to enhance drought tolerance. In particular, ZmCOMT, ZmSNAT, and ZmASMT1 apparently modulated the expression levels of key regulatory genes such as ZmIAA2, ZmIAA23, ZmIAA7, ZmSAUR24, ZmPYL8, and ZmPIF3.1, associated with these hormone pathways. Collectively, endogenous melatonin reinforces drought tolerance by reducing ROS accumulation and reprogramming phytohormone homeostasis through regulation of hormone-related gene expression. Our findings provide important insights into the regulatory mechanisms by which endogenous melatonin enhances drought resistance in crops.},
}

*Melatonin/biosynthesis/genetics
*Zea mays/genetics/metabolism/physiology
*Droughts
*CRISPR-Cas Systems/genetics
*Transcriptome
*Stress, Physiological/genetics
Abscisic Acid/metabolism
Reactive Oxygen Species/metabolism
Gene Expression Regulation, Plant
Mutagenesis
Plant Proteins/genetics/metabolism
Phenotype
Genes, Plant
Gene Expression Profiling

@article {pmid41826557,
year = {2026},
author = {Chao, K and Dietrich, ML and Covey, SC and Momoh, M and Gutt, EG and Sandi, JD and Kamara, MS and Fofanah, IU and Rogers, MM and Kallon, TMPS and Samuels, RJ and Grant, DS and Sabeti, PC and Garry, RF},
title = {Adaptable, quantitative CRISPR/Cas12a-based assay for cytomegalovirus DNA in infant saliva.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826557},
issn = {2045-2322},
mesh = {Humans ; *Cytomegalovirus/genetics/isolation & purification ; *DNA, Viral/genetics/analysis ; *Saliva/virology ; *Cytomegalovirus Infections/diagnosis/virology/genetics ; *CRISPR-Cas Systems/genetics ; Infant ; Sensitivity and Specificity ; Infant, Newborn ; Polymerase Chain Reaction ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Congenital cytomegalovirus (CMV) infection is the leading non-genetic cause of infant hearing loss worldwide, and a significant cause of neurodevelopmental disabilities. Reliance on polymerase chain reaction (PCR) for CMV DNA testing hampers diagnostic and research efforts in low-resource settings and universal screening implementation in high-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) detection and recombinase polymerase amplification (RPA) can be used together for low-cost viral detection. Here we describe an adaptable RPA-Cas12a assay for CMV DNA quantification based on the WHO international standard. Adequate quantification accuracy was achieved with contrived CMV samples but performance with Sierra Leonean infant saliva remains suboptimal. While improved quantification accuracy will require further optimization, our assay achieves screening test requirements, including > 80% sensitivity/specificity, quicker and more economically than PCR. This work highlights RPA-Cas12a-based assays for DNA quantification and suggests a path towards increased congenital CMV screening using PCR and RPA-Cas12a synergistically.},
}

Humans
*Cytomegalovirus/genetics/isolation & purification
*DNA, Viral/genetics/analysis
*Saliva/virology
*Cytomegalovirus Infections/diagnosis/virology/genetics
*CRISPR-Cas Systems/genetics
Infant
Sensitivity and Specificity
Infant, Newborn
Polymerase Chain Reaction
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41826615,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826615},
issn = {2045-2322},
mesh = {*Lipopolysaccharides/biosynthesis/genetics ; *Leptospira interrogans/genetics/metabolism/enzymology ; Animals ; *CRISPR-Cas Systems ; Leptospirosis/microbiology ; Mutagenesis ; *Genes, Essential ; O Antigens/genetics ; *Bacterial Proteins/genetics/metabolism ; Cricetinae ; Hexosyltransferases ; },
abstract = {Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira. Lipopolysaccharide (LPS) is an immunodominant and protective antigen for Leptospira, but its biosynthesis remains poorly understood. In this study, we employed CRISPR/Cas9-non-homologous end-joining and CRISPR-Prime Editing to mutate key genes within the rfb locus of L. interrogans, including those involved in core oligosaccharide assembly, and the biogenesis, polymerization, and ligation of O-antigen. Mutants were successfully generated in LIC11312 (waaF, heptosyltransferase II) and LIC12137 (wcaJ, undecaprenyl-phosphate glycosyltransferase) but yielded only in-frame deletions suggesting their essentiality. Mutants were also successfully generated in LIC12143, a putative O-antigen polymerase, which exhibited truncated LPS that failed to induce acute leptospirosis in hamsters but retained the ability to colonize kidneys. Mutation of LIC_RS09320, an O-antigen ligase, did not display a change in LPS phenotype. Bacterins prepared with either control wild-type or LIC12143 mutant cells conferred complete homologous protection with sterile immunity, though failed to protect against heterologous challenge. These findings confirm LIC12143 as a functional O-antigen polymerase and underscore the challenges in generating knockout mutants to understand LPS biosynthesis in leptospires.},
}

*Lipopolysaccharides/biosynthesis/genetics
*Leptospira interrogans/genetics/metabolism/enzymology
Animals
*CRISPR-Cas Systems
Leptospirosis/microbiology
Mutagenesis
*Genes, Essential
O Antigens/genetics
*Bacterial Proteins/genetics/metabolism
Cricetinae
Hexosyltransferases