@article {pmid41277686,
year = {2025},
author = {Stohr, AM and Hansen, H and Richards, B and Park, H and Goncalves, AG and Agrawal, A and Blenner, M and Chen, W},
title = {Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
doi = {10.1093/nar/gkaf1290},
pmid = {41277686},
issn = {1362-4962},
support = {MCB2317398//National Science Foundation/ ; GM133803/NH/NIH HHS/United States ; //U.S. Department of Defense/ ; P200A210065//GAANN Fellowship Program/ ; },
mesh = {*CRISPR-Cas Systems ; Theophylline/metabolism/pharmacology ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Tryptophan/metabolism/pharmacology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Transcription, Genetic ; *Gene Expression Regulation ; },
abstract = {CRISPR activation is a powerful tool to upregulate a vast array of genes in many different contexts. However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor-actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.},
}

*CRISPR-Cas Systems
Theophylline/metabolism/pharmacology
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Tryptophan/metabolism/pharmacology
*Clustered Regularly Interspaced Short Palindromic Repeats
Transcriptional Activation
Transcription, Genetic
*Gene Expression Regulation

@article {pmid41277071,
year = {2025},
author = {Hotta, M and Inoue, YU and Asami, J and Hoshino, M and Inoue, T},
title = {Generation of a Triple Tag Knock-In Mouse to Visualize Precise Protein Localization Patterns for Type II Classic Cadherins During Brain Development.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {30},
number = {6},
pages = {e70070},
doi = {10.1111/gtc.70070},
pmid = {41277071},
issn = {1365-2443},
support = {NCNP 3-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; NCNP 6-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; },
mesh = {Animals ; *Cadherins/metabolism/genetics ; Mice ; *Brain/metabolism/embryology ; Gene Knock-In Techniques ; Gene Expression Regulation, Developmental ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {Classic cadherin cell-cell adhesion molecules with self-organizing activities play roles in segregating distinct populations of cells at developing brain regions and/or boundaries. However, the protein dynamics of each cadherin subclass in the mouse embryonic brain is poorly described due to the low antigenicity. Here, we generate Cdh6-HA and Cdh8-PA tag knock-in (KI) mice by CRISPR/Cas9-mediated genome editing and establish Cdh6[HA/HA]; Cdh8[PA/PA]; Cdh11[EGFP/EGFP] triple tag KI homo mice with normal viability and fertility. Immunostaining with specific antibodies for these tags reveals differential protein expression profiles almost comparable with mRNA in situ hybridization (ISH) results during embryonic brain development. We can additionally detect considerable levels of immunostaining signals outside the mRNA ISH-positive areas, specifically along the nerve tracts, suggesting physiological accumulation of these type II cadherin proteins along axons. By using super-resolution imaging, we further evaluate cadherin subcellular localization dynamics around the zona limitans intrathalamica to confirm that the prosomere 2/3 compartment boundary at E12.5 is maintained by the distinctive integration of Cdh6 or Cdh11 at apical attachment sites of the ventricular cells. These results highlight the value of the genetic tag KI strategy for proteins with low antigenicity and the functional relevance of type II classic cadherins in brain development.},
}

Animals
*Cadherins/metabolism/genetics
Mice
*Brain/metabolism/embryology
Gene Knock-In Techniques
Gene Expression Regulation, Developmental
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid41276731,
year = {2025},
author = {Eren Eroglu, AE and Toklu, K and Yasa, İ},
title = {Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {253},
pmid = {41276731},
issn = {1438-7948},
}

@article {pmid41276041,
year = {2025},
author = {Khan, MF and Javed, M and Kaur, J and Badwal, AK and Singh, S},
title = {CRISPR-Cas mediated targeting of resistance genes for combating ESKAPE pathogen infections: A Review.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {149180},
doi = {10.1016/j.ijbiomac.2025.149180},
pmid = {41276041},
issn = {1879-0003},
abstract = {Advancements in the treatment of antimicrobial infections have highlighted the importance of the CRISPR-Cas system in targeting resistance genes in bacterial pathogens resistant to conventional drugs. Various CRISPR-Cas techniques, such as CRISPR-Cas9, Cas3, dCas9 and the mini-CRISPR system, have been utilized for this purpose in ESKAPE pathogens. Novel strategies like Associates Toxin Antitoxin and CRISPR-Cas to kill multidrug resistant pathogens-CRISPR-regulated toxin antitoxin module (ATTACK-CreTA) and CRISPR interference refine CRISPR-Cas efficacy. This review explores the mechanism of action of resistance genes (e.g., tetM, ermB, VanA, aph-3, aac3, oxa23, blaNDM etc.) prevalent within these pathogens and highlights the notable achievements of CRISPR-Cas technology in targeting these genes, thereby offering a pathway to sensitize resistant bacteria. This article also discusses various delivery approaches for CRISPR components in pathogens, mainly focusing on engineered bacteriophages, including phagemids, temperate phages and virulent phages. Additionally nanoparticles, bacterial conjugation and natural phages hold promise for administering the CRISPR system inside bacteria. Specific targeting of resistance genes in resistant pathogens via CRISPR-Cas based methods would pave a way for combating ESKAPE pathogen infections by reversing the resistance phenotype.},
}

@article {pmid41273791,
year = {2025},
author = {Espinoza-Erazo, VP and Vela-Chauvin, MG and Collantes-Vela, JC and Zapata-Mena, S and Machado, A},
title = {Biofilms of Salmonella: Implications for Food Safety and Public Health.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1177/15353141251389597},
pmid = {41273791},
issn = {1556-7125},
abstract = {Salmonella enterica is a leading cause of foodborne illness worldwide, responsible for an estimated 93.8 million cases and approximately 155,000 deaths annually, according to the World Health Organization. This foodborne pathogen imposes a significant burden on public health and the global economy. A key factor contributing to the persistence and widespread impact of S. enterica is its potential to form biofilms, which may enhance its survival in clinical, industrial, and agricultural environments, making it a major and ongoing public health concern. Biofilms are structured microbial communities encapsulated in a self-produced extracellular matrix that protects against environmental stressors, disinfectants, and antimicrobial agents. This complex phenotype enables Salmonella to colonize food-contact surfaces, medical devices, and host tissues, hampering efforts to eliminate contamination and control transmission. The poultry industry, a key component of the global food supply, is particularly vulnerable to emerging Salmonella strains with increased virulence, stress tolerance, and disinfectant resistance, making biofilm control a top priority. This review aims to provide an updated and comprehensive overview of the mechanisms involved in Salmonella biofilm formation, its implications for food safety, and recent advances in detection and control strategies. Emerging technologies such as CRISPR-Cas systems are receiving particular attention due to their potential as precise molecular tools for investigating genes implicated in biofilm formation. By integrating current findings, this review underscores the urgent need for novel and effective strategies for biofilm control. It highlights the importance of a One Health approach that links human, animal, and environmental health to address the risks posed by Salmonella biofilms in the food production and public health sectors.},
}

@article {pmid41273435,
year = {2025},
author = {Yu, SM and Li, TT and Fu, BQ and Zhang, NZ},
title = {Molecular diagnosis of Trichinella spp.: current status and future prospects.},
journal = {Parasitology research},
volume = {124},
number = {11},
pages = {136},
pmid = {41273435},
issn = {1432-1955},
support = {2023YFD1801000//National Key Research and Development Program of China/ ; QYXTZX-RKZ2024-03-3//Science and Technology Project of Tibet Autonomous Region/ ; },
mesh = {Animals ; *Trichinellosis/diagnosis/parasitology/veterinary ; *Trichinella/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods/trends ; CRISPR-Cas Systems ; Humans ; },
abstract = {Trichinellosis, a significant parasitic zoonotic disease, poses a risk to public health as well as economic implications for the safety of animal feed. Consumption of raw or undercooked meat containing Trichinella larvae can lead to trichinellosis infection. Many molecular diagnostic methods have been developed to identify Trichinella spp. at muscle or intestine stages. However, no molecular diagnostic technique is currently advised for routine testing on Trichinella infection in food animals, particularly at the early stage of infection. Here, the authors review the development of molecular diagnostic techniques of Trichinella spp., such as PCR, RT-PCR, LAMP, RPA and other methods to detect Trichinella DNA. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) technology holds great promise for diagnostic testing by providing rapid, sensitive and specific methods for detection. Diagnosis of Trichinella spp. based on CRISPR-Cas system may be a promising method meeting the needs of individual testing.},
}

Animals
*Trichinellosis/diagnosis/parasitology/veterinary
*Trichinella/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods/trends
CRISPR-Cas Systems
Humans

@article {pmid41273433,
year = {2025},
author = {Schreiber, D and Yang, R and Guan, X and Schalper, KT and Hou, C and Li, Z and Hegde, P and Liu, C},
title = {3D-Printed CRISPR-based detection system powered by a reusable handwarmer.},
journal = {Biomedical microdevices},
volume = {27},
number = {4},
pages = {53},
pmid = {41273433},
issn = {1572-8781},
support = {U01CA269147/NH/NIH HHS/United States ; UConn Research Excellence Program (REP) award//University of Connecticut/ ; },
mesh = {*Printing, Three-Dimensional ; Humans ; Human papillomavirus 16/genetics ; *CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; DNA, Viral/analysis/genetics ; Equipment Reuse ; },
abstract = {Nucleic acid-based molecular diagnostics are essential for the prevention, early detection, and treatment of cancer and infectious diseases. In this study, we developed a 3D-printed, electricity-free detection system for CRISPR-based nucleic acid detection. To eliminate the need for costly electrical heaters, we developed a reusable heating platform powered by a sodium acetate-based handwarmer. To maintain optimal temperatures for the CRISPR reaction, we designed and fabricated a 3D-printed heatsink filled with docosane wax to regulate the temperature. The fully 3D-printed microfluidic chip integrates finger-activated fluid transport via a 3D-printed flexible blister, a CRISPR reaction chamber, and a lateral flow strip for visual readout. We demonstrated the system's analytical performance by detecting HPV-16 DNA with a sensitivity as low as 1 femtomolar. Additionally, we validated its clinical pilot feasibility using clinical cervical samples, achieving results consistent with standard PCR assays. Overall, this low-cost, reusable, and electricity-free detection system offers a practical solution for point-of-care molecular testing, particularly in resource-limited settings.},
}

*Printing, Three-Dimensional
Humans
Human papillomavirus 16/genetics
*CRISPR-Cas Systems/genetics
*Lab-On-A-Chip Devices
DNA, Viral/analysis/genetics
Equipment Reuse

@article {pmid41273185,
year = {2025},
author = {Verma, A and Kaur, L and Kandoth, PK},
title = {Agrobacterium rhizogenes-Mediated Hairy Root Transformation for Genome Editing in Recalcitrant Legume Lathyrus sativus.},
journal = {Current protocols},
volume = {5},
number = {11},
pages = {e70256},
doi = {10.1002/cpz1.70256},
pmid = {41273185},
issn = {2691-1299},
mesh = {*Lathyrus/genetics ; *Agrobacterium/genetics ; *Plant Roots/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus Support Protocol 1: Escherichia coli competent cell preparation and transformation Support Protocol 2: A. rhizogenes competent cell preparation and transformation Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus Basic Protocol 3: Screening of transgenic hairy root lines Support protocol 3: DNA isolation from L. sativus hairy roots.},
}

*Lathyrus/genetics
*Agrobacterium/genetics
*Plant Roots/genetics
*Gene Editing/methods
*Transformation, Genetic
CRISPR-Cas Systems
Plants, Genetically Modified/genetics

@article {pmid41272318,
year = {2025},
author = {Duan, B and Jin, X and An, X and Xiao, Y and Yang, Q and Zhao, H and Huang, Y and Wang, J and Wang, Q and Du, F and Lu, L and Sun, L and Chen, Z and Zhao, B},
title = {Molecular basis of SAM-AMP synthesis and degradation in the type III-B CRISPR-Cas system.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41272318},
issn = {1552-4469},
support = {2021hwyq36//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Upon sensing nonself target RNA, the CorA-associated type III-B CRISPR-Cas system catalyzes S-adenosyl methionine (SAM) and ATP to synthesize SAM-AMP, which activates the effector CorA and triggers immune responses. SAM-AMP can be degraded by NrN and SAM lyase, potentially deactivating the system. Here we find that the type III-B effector complex from Bacteroides fragilis uses a specific mechanism to recognize nonself target RNA and synthesize SAM-AMP. The 3' anti-tag of nonself target RNA induces conformational changes in the Cmr2 subunit, triggering SAM-AMP synthesis independently of the stalk loop of Cmr3 subunit. SAM-AMP binding induces NrN to transit from an open to a closed conformation, enabling hydrolysis of the 3'-5' phosphodiester bond. SAM lyase forms a triangular trimer that specifically degrades SAM-AMP into 5'-methylthioadenosine-AMP and homoserine lactone. These findings unveil unique mechanisms for SAM-AMP synthesis and degradation and provide deeper insights into the molecular basis of type III CRISPR-Cas signaling.},
}

@article {pmid41271814,
year = {2025},
author = {Ueno, R and Ito, S and Oyama, T},
title = {A CRISPR/Cas9-induced restoration of bioluminescence reporter system for single-cell gene expression analysis in plants.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41271},
pmid = {41271814},
issn = {2045-2322},
support = {JPMJSP2110//Japan Science and Technology Agency/ ; JPMJAL1108//Japan Science and Technology Agency/ ; JP20K06342//Japan Society for the Promotion of Science/ ; 17KT0022//Japan Society for the Promotion of Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics/metabolism ; *Single-Cell Analysis/methods ; *Genes, Reporter ; Plants, Genetically Modified/genetics ; *Luminescent Measurements/methods ; *Gene Expression Regulation, Plant ; Luciferases/genetics/metabolism ; Single-Cell Gene Expression Analysis ; },
abstract = {Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression, although bioluminescence could fluctuate according to variation in physiological factors associated with the luciferase reaction. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis. In this method, the enzymatic activity of an inactive luciferase mutant, LUC40Ins26bp, which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp, which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.},
}

*CRISPR-Cas Systems/genetics
*Arabidopsis/genetics/metabolism
*Single-Cell Analysis/methods
*Genes, Reporter
Plants, Genetically Modified/genetics
*Luminescent Measurements/methods
*Gene Expression Regulation, Plant
Luciferases/genetics/metabolism
Single-Cell Gene Expression Analysis

@article {pmid41271724,
year = {2025},
author = {Elsharkasy, OM and Hegeman, CV and Driedonks, TAP and Liang, X and Lansweers, I and Cotugno, OL and de Groot, IY and de Wit, ZEMNJ and Garcia-Guerra, A and Moorman, NJA and Boonstra, SH and Bosman, EDC and Lefferts, JW and de Voogt, WS and François, JJ and van Wesel, ACW and El Andaloussi, S and Schiffelers, RM and Kooijmans, SAA and Mastrobattista, E and Vader, P and de Jong, OG},
title = {A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10309},
pmid = {41271724},
issn = {2041-1723},
support = {VI.Veni.192.174//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; *Aptamers, Nucleotide/metabolism/genetics ; Humans ; *Gene Editing/methods ; Ultraviolet Rays ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains a major hurdle in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles that play an important role in intercellular communication, and have an innate capability of intercellular transfer of biological cargos, including proteins and RNA. Here, we present a versatile, modular strategy for EV-mediated loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins fused to EV-enriched proteins to MS2 aptamers incorporated into guide RNAs, in combination with a UV-activated photocleavable linker domain, PhoCl. Moreover, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e. Taken together, we describe a robust, modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering and transcriptional regulation.},
}

*CRISPR-Cas Systems/genetics
*Extracellular Vesicles/metabolism
*Aptamers, Nucleotide/metabolism/genetics
Humans
*Gene Editing/methods
Ultraviolet Rays
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells

@article {pmid41271690,
year = {2025},
author = {Huang, Z and Dong, Y and Yang, Y and Han, X and Wang, F and Lyon, CJ and Ding, S and Peng, Y and Zhang, G and Hu, C and Huang, H and Yang, L and Zhao, G and Fan, XY and Lu, S and Hu, T and Wang, J},
title = {Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10286},
pmid = {41271690},
issn = {2041-1723},
support = {31922046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pandemics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; Sensitivity and Specificity ; Temperature ; },
abstract = {The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Pandemics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Point-of-Care Systems
Sensitivity and Specificity
Temperature

@article {pmid41271362,
year = {2025},
author = {Rahman, MA and Akter, S and Ashrafudoulla, M and Jung, SJ and Rapak, MT and Ha, SD},
title = {CRISPR-Cas systems as emerging tools for precision biofilm control for food safety: Mechanisms and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {222},
number = {Pt 2},
pages = {117803},
doi = {10.1016/j.foodres.2025.117803},
pmid = {41271362},
issn = {1873-7145},
mesh = {*Biofilms/growth & development ; *CRISPR-Cas Systems ; *Food Safety/methods ; Gene Editing/methods ; *Food Microbiology/methods ; },
abstract = {Biofilms on food-contact surfaces pose persistent challenges to sanitation, safety, and product quality within food processing. Traditional cleaning methods and broad-spectrum antimicrobials often fail to disrupt the resilient matrix and multispecies communities characteristic of these biofilms. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems offer a transformative approach to enhancing food safety, enabling precise modulation of microbial gene networks with applications in diagnostics, programmable sanitation, and targeted microbial control. This review synthesizes recent advances in CRISPR-Cas technology, encompassing Cas9/Cas12-based gene editing, Cas13-mediated RNA targeting, and dead Cas9 (dCas9)-based transcriptional regulation (CRISPR interference/activation, CRISPRi/a), and evaluates their relevance to biofilm prevention and eradication in food environments. We critically assess delivery platforms, including plasmids, nanocarriers, phagemids, and conjugative systems, for their efficiency in complex biofilm settings. The review highlights innovations such as multiplexed repression of redundant pathways, activation of latent antibiofilm functions. These genetic strategies are increasingly being integrated with omics-based analytics (e.g., transcriptomics, proteomics, metabolomics) to reveal systems-level cellular responses and regulatory shifts triggered by biofilm-targeted interventions. We also address the practical limitations, such as delivery barriers, off-target effects, regulatory hurdles, and ethical considerations specific to food applications. Ultimately, we propose a framework for translating CRISPR-Cas technology into scalable, safety-compliant tools for precision control of biofilms in food processing environments. This review aims to guide future research and inform stakeholders on leveraging CRISPR-Cas technology for safe, sustainable, and targeted management of food-associated biofilms.},
}

*Biofilms/growth & development
*CRISPR-Cas Systems
*Food Safety/methods
Gene Editing/methods
*Food Microbiology/methods

@article {pmid41202983,
year = {2026},
author = {Hocq, R and Chartier, G and Lopes Ferreira, N and Wasels, F},
title = {CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {165-169},
doi = {10.1016/j.jbiotec.2025.11.002},
pmid = {41202983},
issn = {1873-4863},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium beijerinckii/genetics/drug effects ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Listeria monocytogenes/genetics ; Genome, Bacterial ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Clostridium beijerinckii/genetics/drug effects
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Listeria monocytogenes/genetics
Genome, Bacterial
CRISPR-Associated Protein 9/genetics

@article {pmid41202696,
year = {2025},
author = {Ito, S and Nakamura, K and Murata, K and Nakajima, R and Kanou, M and Koketsu, M and Yamana, K and Yamanouchi, K and Ueda, H},
title = {Generation of Cre/LoxP-mediated extracellular TurboID knock-in rats with CRISPR/Cas9 system.},
journal = {Biochemical and biophysical research communications},
volume = {791},
number = {},
pages = {152898},
doi = {10.1016/j.bbrc.2025.152898},
pmid = {41202696},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Rats ; *Integrases/metabolism/genetics ; Biotinylation ; Cell Membrane/metabolism ; },
abstract = {The plasma membrane plays a central role in regulating signal transduction from the extracellular environment to the intracellular space and represents a major site of drug targeting. Proximity-dependent biotinylation with biotin ligases such as BioID and its derivatives, including TurboID and AirID, enables identification of novel protein‒protein interactions by fusion to a target protein. In vivo application of biotin ligases for cell membrane analysis has been attempted; however, methods for expressing biotin ligases on the target cell membrane remain largely limited to viral vector delivery. In this study, we inserted a Cre-dependent TurboID expression cassette into the rat genome to express TurboID on the cell surface via the CRISPR/Cas9 system. We generated TurboID knock-in (KI) rats that express TurboID on the cell surface, enabling biotinylation of extracellular proteins. The TurboID KI rats thus provide a valuable model for in vivo analysis of cell-surface molecules and may facilitate identification of novel drug targets or antigens.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Rats
*Integrases/metabolism/genetics
Biotinylation
Cell Membrane/metabolism

@article {pmid41130365,
year = {2026},
author = {Zou, M and Tao, Y and Shi, B and Xu, R and Zhu, D and Li, Y and Han, R and Wang, R},
title = {CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {67-76},
doi = {10.1016/j.jbiotec.2025.10.006},
pmid = {41130365},
issn = {1873-4863},
mesh = {*Halomonas/genetics/metabolism ; *Amino Acids, Diamino/metabolism/biosynthesis/genetics ; Metabolomics/methods ; Gene Deletion ; *CRISPR-Cas Systems/genetics ; Betaine/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.},
}

*Halomonas/genetics/metabolism
*Amino Acids, Diamino/metabolism/biosynthesis/genetics
Metabolomics/methods
Gene Deletion
*CRISPR-Cas Systems/genetics
Betaine/metabolism
Bacterial Proteins/genetics/metabolism

@article {pmid41048015,
year = {2025},
author = {Li, Y and Zhao, W and Wu, Y and Li, R and Zhang, J and Xie, H and Zhang, K and Li, J},
title = {In vivo CRISPR biosensing.},
journal = {Chemical Society reviews},
volume = {54},
number = {23},
pages = {10977-11016},
doi = {10.1039/d5cs00921a},
pmid = {41048015},
issn = {1460-4744},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/analysis ; Gene Editing ; },
abstract = {In vivo biosensing is essential for real-time monitoring of biological processes and disease progression within living organisms. Leveraging the programmable specificity and multifunctionality of CRISPR effectors, in vivo CRISPR-based biosensing has emerged as a powerful tool for highly sensitive and target-specific detection in complex physiological environments. This review presents the fundamental principles, design strategies, and bioanalytical applications of these advanced sensors, focusing on three key approaches: CRISPR-mediated highly efficient in vivo sequence recognition, CRISPR-driven trans-cleavage activity for signal amplification, and the use of base editors and prime editors for sensing-coupled genetic modulation. Critical design parameters-including delivery strategies, intracellular dynamics, and signal amplification mechanisms-are discussed in detail. We further highlight a broad range of applications, including in vivo DNA/RNA imaging, quantification of proteins and small molecules, gene-controlled drug release, dynamic signal recording, environmental response sensing, and lineage tracing in embryogenesis and tumor progression. The current challenges and outlining future directions are also discussed, underscoring the transformative potential of in vivo CRISPR biosensing in both fundamental biology and clinical translation.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics/analysis
Gene Editing

@article {pmid40555270,
year = {2025},
author = {Liang, L and Yang, Y and Jacqueline Elise, F and Yu, J and Yin, X and Lu, G and Chen, B and Xing, J},
title = {Potential Applications of the CRISPR-Cas9 System for Research and Treatment of Osteoarthritis.},
journal = {Zeitschrift fur Orthopadie und Unfallchirurgie},
volume = {163},
number = {6},
pages = {510-517},
doi = {10.1055/a-2616-0819},
pmid = {40555270},
issn = {1864-6743},
support = {2308085QH292//Anhui Natural Science Foundation/ ; 82305280//This research did receive grant from National Natural Science Foundation of China/ ; },
mesh = {*Osteoarthritis/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {Osteoarthritis is a common degenerative disease of joint cartilage that affects millions of people in the world, especially the elderly. Progression of osteoarthritis is associated with a plethora of genetic and non-genetic factors. The CRISPR/Cas9 system is emerging as a powerful tool for genome engineering and has remarkable potential for guiding further research into osteoarthritis and may be a viable means for treating the disease. This review discusses existing and potential applications of the CRISPR/Cas9 system in osteoarthritis studies and treatments. Firstly, we briefly summarize the current status and mechanism of this technology. Next, we focus on the latest advances in the application of CRISPR/Cas9 system in elucidating the contributions of various factors to the pathogenesis of osteoarthritis as demonstrated through in vitro studies and animal models. Finally, we provide our perspective on the direction and challenges of studying and treating osteoarthritis with CRISPR/Cas9.},
}

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

@article {pmid41270733,
year = {2025},
author = {Wei, Z and Lan, Y and Meng, L and Wang, H and Li, L and Li, Y and Zhang, N and Lu, R and Cui, Z and Song, Y and Wang, Y and Li, Y and Yue, Z and Fan, G and Li, Q and Gu, Y and Liu, S and Qian, PY and Meng, L and Shao, C},
title = {Hologenomic insights into the molecular adaptation of deep-sea coral Bathypathes pseudoalternata.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.10.020},
pmid = {41270733},
issn = {1934-6069},
abstract = {Deep-sea coral ecosystems support biodiversity and nutrient cycling through interactions with symbionts. However, their molecular mechanisms remain unexplored. Here, hologenomic analyses of Bathypathes pseudoalternata are applied to uncover molecular adaptations underpinning host-symbiont interactions. Genomic evidence reveals that B. pseudoalternata exhibits adaptations in nutrient transport, immune response, and lysosomal digestion, reflecting its genomic adjustments for a stable symbiosis. Candidatus Nitrosopumilus bathypathes (78.43% ± 3.65%) is inferred to oxidize host-derived ammonia to synthesize amino acids and vitamins to provision the host. The presence of CRISPR-Cas and restriction-modification (R-M) systems suggests that Ca. Bathyplasma bathypathes and Ca. Thalassoplasma bathypathes (10.68% ± 2.99%) may protect the host from viral infections. Ca. Bathybacter bathypathes (8.39% ± 1.53%) is hypothesized to synthesize heme, lipoic acid, and glutathione, which serve dual functions as antioxidants and nutrients. These findings collectively provide insights into how the hologenome contributes to the survival of B. pseudoalternata in the extreme environment.},
}

@article {pmid41270608,
year = {2025},
author = {Zhou, Y and Zhai, J and Chen, H and Qu, Y and Fang, Z and Chen, B and Bao, Z and Chen, D},
title = {Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129139},
doi = {10.1016/j.talanta.2025.129139},
pmid = {41270608},
issn = {1873-3573},
abstract = {Lead (Pb[2+]) poses serious risks to health and ecosystems, necessitating rapid, ultrasensitive detection. CRISPR/Cas12a systems offers exceptional specificity and intrinsic signal amplification. Nevertheless, their adaptation to Pb[2+] is hindered by the lack of programmable interfaces to convert small-molecule binding into Cas12a activation. Herein, we report a DNAzyme-Switched G-quadruplex-locked CRISPR (Dz-SiG CRISPR) strategy, enabling ultrasensitive, femtomolar-level detection of Pb[2+]. A RNA G-quadruplex (RG4) structure is conjugated to the 5' end of the crRNA, serving as a conformational lock that suppresses Cas12a's trans-cleavage activity. Upon Pb[2+] binding, the GR-5 DNAzyme catalyzes a hydrolytic cleavage that acts as a molecular switch, releasing the RG4 domain and unleashing active crRNA to trigger Cas12a-mediated cleavage of a fluorogenic reporter, yielding a sharp "off-to-on" fluorescent signal. The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for Pb[2+] and demonstrates outstanding performance in real water and soil samples, with recovery rates ranging from 94.44 % to 99.03 %. The assay can be completed within 30 min, making it highly suitable for rapid on-site lead ion detection. Importantly, the modular Dz-SiG CRISPR framework can be readily reprogrammed for other small molecules by simply substituting the DNAzyme module, offering a generalizable strategy for rapid, ultrasensitive environmental monitoring.},
}

@article {pmid41202468,
year = {2025},
author = {Zingarelli, F and Nanni, J and Cristiano, G and Zannoni, L and Curti, A},
title = {CRISPR-Cas9 in acute myeloid leukaemia: Current state-of-art and future perspectives.},
journal = {Current opinion in pharmacology},
volume = {85},
number = {},
pages = {102582},
doi = {10.1016/j.coph.2025.102582},
pmid = {41202468},
issn = {1471-4973},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/therapy/diagnosis ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 gene editing technology has gained attention as a new, reliable and manageable tool for the treatment of previously incurable monogenic diseases. Besides exciting results in this setting, ethical, safety and crucial technical issues have not been fully clarified. More importantly, the role of this potent editing tool in the context of a genetically complex and heterogeneous hematologic malignancy such as acute myeloid leukemia (AML) has not yet been defined to date. In this review we aim to summarize and exploring the ultimate CRISPR-cas9 based strategies for diagnosis, risk stratification and treatment in the context of AML.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/therapy/diagnosis
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Genetic Therapy/methods

@article {pmid41177337,
year = {2025},
author = {Liu, B and Li, Y and Yang, Z and Wu, J and Jiang, Y and Zhao, L and Ge, J},
title = {A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology.},
journal = {Journal of microbiological methods},
volume = {239},
number = {},
pages = {107314},
doi = {10.1016/j.mimet.2025.107314},
pmid = {41177337},
issn = {1872-8359},
mesh = {Animals ; Dogs ; *CRISPR-Cas Systems/genetics ; *Adenoviridae Infections/diagnosis/veterinary/virology ; *Adenoviruses, Canine/isolation & purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Dog Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10[2] copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.},
}

Animals
Dogs
*CRISPR-Cas Systems/genetics
*Adenoviridae Infections/diagnosis/veterinary/virology
*Adenoviruses, Canine/isolation & purification/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Dog Diseases/diagnosis/virology
*Molecular Diagnostic Techniques/methods

@article {pmid41167358,
year = {2025},
author = {Rahangdale, S and Vishwakarma, A and Chauhan, R and Singh, S and Singh, PK},
title = {Engineered sgRNA captures single-stranded donor template and delivers at the DSB site to enhance HDR.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148614},
doi = {10.1016/j.ijbiomac.2025.148614},
pmid = {41167358},
issn = {1879-0003},
mesh = {Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *Recombinational DNA Repair ; *DNA, Single-Stranded/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has revolutionized genome modification through precise editing across a wide range of organisms. Yeast supports efficient genome editing via plasmid-based Cas9-gRNA expression, while higher eukaryotes often require genome-integrated cassettes or RNP delivery. In this study, we engineered CRISPR components to enhance nuclear targeting and editing efficiency. We demonstrated the proof of concept in Saccharomyces cerevisiae using its CAN1 locus. We developed a dual-host compatible vector, encoding Cas9 nuclease fused with three nuclear localization signals (Cas9-3xNLS). The recombinant protein, expressed in E. coli and purified on a Ni-NTA column, showed DNA cleavage in an in vitro assay. Genome editing efficacy of Cas9-3xNLS was demonstrated in S. cerevisiae AH109 strain. Further, we engineered sgRNAs by extending their ends to facilitate the annealing to ssODN. We synthesized ssODNs having a complementary sequence either at 3' or 5' to anchor with sgRNAs. sgRNAs (unmodified and end extended) and ssODNs were introduced into yeast in various combinations. sgRNA with a 3' ssDNA-anchoring motif and ssODN representing the antisense strand of the target gene with sgRNA complementary motif at the 5' end (free homology arm at 3') improved HDR efficiency significantly. This combination yielded about a 1.64-fold increase in canavanine-resistant colonies as compared to the control via precise insertion of a stop codon. In contrast, extension of sgRNA at the 5' end did not show any advantage. This approach is flexible and easy to use and has the potential to enhance homology-directed repair in diverse organisms.},
}

Saccharomyces cerevisiae/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
*DNA Breaks, Double-Stranded
*Recombinational DNA Repair
*DNA, Single-Stranded/genetics
Streptococcus pyogenes/genetics

@article {pmid41093192,
year = {2025},
author = {Chou, SJ and Wang, CH and Chang, YL and Fang, WC and Hwang, DK and Hsiao, YJ and Luo, YH and Lo, WL and Viet, NQ and Tang, KY and Lan, YT and Hsu, CC and Chen, SJ and Lin, TC and Yang, YP and Chiou, SH},
title = {Dual delivery of supramolecular nanoparticle-carried minicircle donor DNA with Cas9/gRNA improved HITI knock-in efficiency in X-linked juvenile retinoschisis.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148300},
doi = {10.1016/j.ijbiomac.2025.148300},
pmid = {41093192},
issn = {1879-0003},
mesh = {Humans ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Retinoschisis/genetics/therapy ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA, Circular/genetics ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/genetics ; Genetic Therapy/methods ; HEK293 Cells ; Eye Proteins/genetics ; Plasmids/genetics ; },
abstract = {X-linked retinoschisis (XLRS) is a hereditary mutation of the RS1 gene and is characterized by early-onset maculopathy with severe visual impairment. Current gene therapy utilizing CRISPR-associated protein 9 (Cas9) is ongoing; however, the optimization of nonviral/nanoparticle CRISPR/Cas9-based therapeutics for gene delivery into nondivided retinal neurons remains undetermined. Minicircles DNA, a circular DNA molecule lacking bacterial backbone sequences, has gained recognition for improving transfection efficiency and biosafety. Here, we developed a supramolecular nanoparticle (SMNP)-editing platform in which SMNPs carrying CRISPR/Cas9 integrated minicircle donor DNA (mc dDNA) to achieve highly efficient and precise gene knock-in. To increase the efficiency of RS1 gene knock-in, we replaced the conventional mc dDNA with a single flanking-Cas9/cut site. Furthermore, using homology-independent targeted integration (HITI) as an editing-nondivided cell strategy, SMNP-carried CRISPR/Cas9 could effectively facilitate the dual delivery of mc-RS1/GFP dDNA and Cas9/gRNA plasmids. Compared with the delivery of the Cas9/gRNA plasmid alone (~20 %), the delivery of mc-RS1/GFP dDNA via SMNPs had significantly higher transfection efficiency (90 %). Further flow cytometry analysis revealed that 5.99 % of the FACS-positive cells were detected in the mc-RS1/GFP dDNA group, markedly exceeding the 2.21 % with long-term expression in the conventional dDNA group. Moreover, when XLRS/iPSC-derived retinal neuron organoids were used as a patient-based disease model, compared with conventional plasmid-based delivery, robust RS1 expression with integration sustained transgene expression in XLRS/iPSC-derived retinal organoids. Collectively, these findings indicated that SMNP-mediated dual delivery of the Cas9/gRNA plasmid and mc-RS1/GFP dDNA substantially enhanced RS1-targeted integration with long-term transgene expression, providing safer and effective gene therapy for the treatment of XLRS.},
}

Humans
*Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
*Retinoschisis/genetics/therapy
*RNA, Guide, CRISPR-Cas Systems/genetics
*DNA, Circular/genetics
Gene Editing/methods
*Gene Knock-In Techniques/methods
Gene Transfer Techniques
CRISPR-Associated Protein 9/genetics
Genetic Therapy/methods
HEK293 Cells
Eye Proteins/genetics
Plasmids/genetics

@article {pmid41270157,
year = {2025},
author = {Li, Y and Wu, Y and Zheng, Z and Wu, Y and Zhang, Y and Zhang, J and Quan, F and Zhao, W and Xu, R and Li, Y and Gao, H and Zhang, K},
title = {Renal clearable CRISPR nanosensor targeting mitochondrial DNA mutation for noninvasive monitoring of tumor progression and metastasis.},
journal = {Science advances},
volume = {11},
number = {47},
pages = {eadz4594},
pmid = {41270157},
issn = {2375-2548},
mesh = {*DNA, Mitochondrial/genetics ; *Mutation ; Humans ; *CRISPR-Cas Systems ; Animals ; Disease Progression ; Neoplasm Metastasis ; Mice ; Biomarkers, Tumor/genetics/urine ; Cell Line, Tumor ; *Biosensing Techniques/methods ; Mitochondria/genetics ; Lung Neoplasms/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are emerging as important molecular features of tumorigenesis. Liquid biopsies, involving analysis of cell-free mtDNA, enable early cancer detection but suffer from low sensitivity due to scarce analytes. Here, we developed a CRISPR/Cas12a-mediated urinary biomarker, termed CasUber, for in vivo monitoring of tumor progression and metastasis. Our results demonstrate that CasUber can deliver a CRISPR detection system into tumor cell mitochondria, leverage the single-nucleotide variant recognition ability and trans-cleavage activity of Cas12a to convert tumor-specific mtDNA mutations into renal-clearable fluorescent biomarkers, and exocytosed along with the natural efflux pathway of damaged mtDNA. As a result, CasUber enables discrimination of ultrasmall tumor lesions (~1 cubic millimeter) and detection of lung tumor nodules earlier than bioluminescence imaging in a blood-lung metastasis model. This renal clearable nanosensor allows in situ recognition of specific gene mutation to generate amplified signals, overcoming the limitation of low mtDNA abundance and enabling noninvasive and ultrasensitive monitoring of tumor progression and metastasis via a simple urine test.},
}

*DNA, Mitochondrial/genetics
*Mutation
Humans
*CRISPR-Cas Systems
Animals
Disease Progression
Neoplasm Metastasis
Mice
Biomarkers, Tumor/genetics/urine
Cell Line, Tumor
*Biosensing Techniques/methods
Mitochondria/genetics
Lung Neoplasms/genetics

@article {pmid41266743,
year = {2025},
author = {Zhang, W and Shi, J and Wang, B and Qu, H and Wu, X and Wang, X},
title = {An integrated Aptamer-CRISPR-Cas12a method for rapid and sensitive detection of carbendazim.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41119},
pmid = {41266743},
issn = {2045-2322},
support = {22A310016//Henan Province Higher Education Key Scientific Research Project/ ; },
mesh = {*Carbamates/analysis ; *Aptamers, Nucleotide/genetics/chemistry ; *CRISPR-Cas Systems ; *Benzimidazoles/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; *Pesticide Residues/analysis ; Reproducibility of Results ; },
abstract = {Concerns over carbendazim (CBZ) pesticide residues in agricultural products and medicinal herbs have intensified due to their potential health and environmental risks. While existing detection techniques offer distinct advantages, they are often limited by complex procedures, specialized equipment, and high costs. To address these challenges, we developed a novel Aptamer-CRISPR/Cas12a assay, which combines the specificity of aptamers with the high sensitivity and precision of the CRISPR/Cas12a system. This assay achieves a linear detection range of 10-5000 ng/mL (R[2] = 0.9639 at 15 min and R[2] = 0.9774 at 30 min) and a limit of detection (LOD) of 10 ng/mL. In real samples, the average recovery rate of CBZ ranges from 92.10% to 102.86%, demonstrating robust accuracy and reliability. Notably, the method is user-friendly, requires minimal equipment, and delivers results in about 40 min, making it suitable for field applications. Furthermore, the crRNA serves as a universal sequence, enabling the detection of different targets by simply replacing the aptamer and complementary strand, while keeping the CRISPR/Cas12a system intact. This streamlined approach enhances flexibility and broad applicability. In conclusion, the Aptamer-CRISPR/Cas12a assay offers a practical solution for monitoring agricultural products, Chinese herbal medicine, and environmental safety.},
}

*Carbamates/analysis
*Aptamers, Nucleotide/genetics/chemistry
*CRISPR-Cas Systems
*Benzimidazoles/analysis
Limit of Detection
*Biosensing Techniques/methods
*Pesticide Residues/analysis
Reproducibility of Results

@article {pmid41266199,
year = {2025},
author = {Wu, X and Wang, M and Luo, S and Zhou, Z and Wang, Y and Du, G and Chen, J and Liu, X},
title = {Dual enhancement of mycoprotein nutrition and sustainability via CRISPR-mediated metabolic engineering of Fusarium venenatum.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.09.016},
pmid = {41266199},
issn = {1879-3096},
abstract = {Mycoprotein (MP) production represents a promising environmentally sustainable strategy to address global protein deficit. To enhance the nutritional profile and production efficiency of MP, we employed CRISPR/Cas9-mediated scarless gene knockout and obtained a Fusarium venenatum strain (designated FCPD), which exhibited a 32.9% increase in essential amino acid index (EAAI) through targeted truncation of competitive metabolic pathways and regulation of amino acid metabolism or biosynthesis. FCPD achieved a 44.3% reduction in substrate consumption while improving MP production rate by 88.4% compared with the wild type (WT) strain. The cradle-to-gate life cycle assessment (LCA) shows that FCPD could reduce environmental impacts such as global warming potential (GWP) by 4-61.3% under production scenarios in six representative countries. Comparative environmental performance demonstrated the superiority of FCPD-MP over cell-cultured meat and chicken meat. These findings establish CRISPR/Cas technology and metabolic engineering as the dual-purpose tool for both nutritional enhancement and environmental impact mitigation in alternative protein production.},
}

@article {pmid41265250,
year = {2025},
author = {Ramachandran, H and Becker, A and Dobner, J and Hildebrandt, B and Distelmaier, F and Rossi, A and Anand, R},
title = {CRISPR/Cas9-mediated editing of MIC13 in human induced pluripotent stem cells: A model for mitochondrial hepato-encephalopathy.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103870},
doi = {10.1016/j.scr.2025.103870},
pmid = {41265250},
issn = {1876-7753},
abstract = {MIC13 is essential for cristae formation and functions as a key component of the large mitochondrial multi subunit MICOS complex. Mutations in MIC13 causes severe mitochondrial disease called mitochondrial hepato-encephalopathy. In this study, we describe the generation of a human induced pluripotent stem cell (iPSC) line carrying a patient-specific MIC13 mutation, introduced using a CRISPR/Cas knock-in approach. The resulting iPSC line will provide a valuable model to study the pediatric severe mitochondrial disease and to determine the pathological mechanisms as well as to facilitate the identification of potential therapeutic targets in the future.},
}

@article {pmid41264989,
year = {2025},
author = {Liu, X and Yang, M and Sun, D and Lu, C and Ma, Y and Jiang, Y and Ouyang, R and Miao, Y},
title = {Integrating amplification strategies and functional nanomaterials for advanced electrochemical biosensing of MicroRNA.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {168},
number = {},
pages = {109170},
doi = {10.1016/j.bioelechem.2025.109170},
pmid = {41264989},
issn = {1878-562X},
abstract = {MicroRNAs (miRNAs) are crucial disease biomarkers, yet their short length, low abundance, and high sequence homology pose significant challenges for sensitive detection. Electrochemical biosensing presents a promising alternative, though effective signal amplification remains essential. This review summarizes recent advances in amplification strategies for electrochemical miRNA detection, covering nucleic acid-based techniques-such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and catalytic hairpin assembly (CHA)-as well as nanomaterial-assisted approaches using metal-organic frameworks and transition metal dichalcogenides. Key mechanisms, advantages, and limitations of each method are discussed, along with performance metrics (e.g., detection limit and linear range) and emerging hybrid systems like RCA-CRISPR/Cas. Current challenges, including probe complexity and nanomaterial aggregation, are also addressed. Finally, the review highlights future directions involving multi-mechanism integration and clinical translation, offering insights for the development of highly sensitive and reliable electrochemical biosensors to advance precision medicine.},
}

@article {pmid41233602,
year = {2025},
author = {Strefeler, A and Baker, ZN and Chollet, S and Foged, MM and Guerra, RM and Ivanisevic, J and Gallart-Ayala, H and Pagliarini, DJ and Jourdain, AA},
title = {Uridine-sensitized screening identifies demethoxy-coenzyme Q and NUDT5 as regulators of nucleotide synthesis.},
journal = {Nature metabolism},
volume = {7},
number = {11},
pages = {2221-2235},
pmid = {41233602},
issn = {2522-5812},
support = {310030_200796//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Pyrophosphatases/metabolism/genetics ; Humans ; *Nucleotides/biosynthesis ; *Uridine/metabolism/pharmacology ; Phosphoribosyl Pyrophosphate/metabolism ; CRISPR-Cas Systems ; Pyrimidines/biosynthesis ; },
abstract = {Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited therapeutic target against cancer, autoinflammatory disorders and viral infections. However, regulation of nucleotide metabolism remains incompletely understood. Here, we reveal regulators of de novo pyrimidine synthesis. Using uridine-sensitized CRISPR-Cas9 screening, we show that coenzyme Q (CoQ) is dispensable for pyrimidine synthesis, in the presence of the demethoxy-CoQ intermediate as alternative electron acceptor. We further report that the ADP-ribose pyrophosphatase NUDT5 directly binds PPAT, the rate-limiting enzyme in purine synthesis, which inhibits its activity and preserves the phosphoribosyl pyrophosphate (PRPP) pool. In the absence of NUDT5, hyperactive purine synthesis exhausts the PRPP pool at the expense of pyrimidine synthesis, which promotes resistance to purine and pyrimidine nucleobase analogues. Of note, the interaction between NUDT5 and PPAT is disrupted by PRPP, highlighting an intricate allosteric regulation. Overall, our findings reveal a fundamental mechanism of nucleotide balance and position NUDT5 as a regulator of nucleobase analogue metabolism.},
}

*Pyrophosphatases/metabolism/genetics
Humans
*Nucleotides/biosynthesis
*Uridine/metabolism/pharmacology
Phosphoribosyl Pyrophosphate/metabolism
CRISPR-Cas Systems
Pyrimidines/biosynthesis

@article {pmid40992601,
year = {2026},
author = {Demirayak, PS and Akay Sazaklioglu, S},
title = {CRISPR for detection of drug resistance genes.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120626},
doi = {10.1016/j.cca.2025.120626},
pmid = {40992601},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Drug Resistance/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Resistance to antibiotics, anticancer, antiviral, and antiparasitic drugs has become one of the greatest threats to modern medicine, seriously straining global health systems. Antimicrobial resistance threatens the integrity of the health system by reducing the effectiveness of treatment protocols such as chemotherapy, organ transplantation, and major surgical interventions. In this case, not only the development of new drugs but also the rapid, sensitive, and specific detection of resistant microorganisms and genetic markers is of vital importance. Therefore, the need for more innovative diagnostic approaches suitable for field applications is increasing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based molecular diagnostic systems developed in recent years stand out as strong candidates that can fill the gap in this area. Thanks to their ability to recognize and target specific DNA or RNA sequences with high specificity, CRISPR systems enable rapid and sensitive detection of drug resistance genes. Various CRISPR effector proteins, such as Cas9, Cas12, and Cas13, have the potential to revolutionize diagnostic technologies due to their ability to both target-specifically cut and generate signals. This review will focus on the application of CRISPR technology for detecting drug resistance genes. In addition, the sensitivity, specificity, application areas, and technical challenges of the systems will be discussed through literature examples of current applications. The review aims to synthesize scientific developments in this field by examining how CRISPR-based diagnostic approaches can play a role in the global fight against drug resistance and to provide a guiding resource for future research.},
}

Humans
*CRISPR-Cas Systems/genetics
*Drug Resistance/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40992599,
year = {2026},
author = {Jiang, T and Zhang, C and Wang, D and Guo, Z and Guo, Y and Liu, H and Wang, Z},
title = {Rapid molecular diagnostic method for Gardnerella vaginalis based on CRISPR-Cas12a and recombinase-aided amplification (RAA).},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120625},
doi = {10.1016/j.cca.2025.120625},
pmid = {40992599},
issn = {1873-3492},
mesh = {*Gardnerella vaginalis/genetics/isolation & purification ; Humans ; Female ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Molecular Diagnostic Techniques/methods ; *Vaginosis, Bacterial/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Imbalance of the vaginal microbiota, particularly the overgrowth of Gardnerella vaginalis, is the primary cause of bacterial vaginosis (BV), which poses a significant threat to women's reproductive health. Therefore, early and rapid diagnosis of BV is crucial. Current laboratory diagnostic methods for BV mainly rely on Amsel's clinical criteria, bacterial culture, and PCR techniques. However, these methods have notable limitations: Amsel's criteria are subject to operator subjectivity, culture methods are time-consuming and require specialized expertise, while PCR necessitates expensive instrumentation. These constraints hinder their widespread clinical application. To address this issue, developing a highly accurate and low-cost molecular diagnostic method holds significant clinical value for BV detection. In recent years, recombinase-aided amplification (RAA) and CRISPR-Cas12a gene-editing technologies have achieved groundbreaking progress in nucleic acid detection. This study innovatively integrates RAA isothermal amplification with CRISPR-Cas12a detection to successfully establish a rapid nucleic acid detection platform for Gardnerella vaginalis. Experimental results demonstrate that this platform achieves a detection sensitivity of 10 copies/mL for Gardnerella vaginalis genomic DNA, with no cross-reactivity against other common reproductive tract pathogens. In validation tests using 44 clinical vaginal swab samples, the platform showed a 100.00 % positive agreement rate compared to qPCR. These findings confirm that the CRISPR-Cas12a-based detection platform exhibits excellent specificity, sensitivity, and reliability, serving as an effective tool for monitoring Gardnerella vaginalis colonization levels. This approach provides a novel molecular diagnostic solution for early BV screening and prevention.},
}

*Gardnerella vaginalis/genetics/isolation & purification
Humans
Female
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Molecular Diagnostic Techniques/methods
*Vaginosis, Bacterial/diagnosis/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41264695,
year = {2025},
author = {Rönspies, M and Khosravi, S and Helia, O and Valisi, A and Fajkus, J and Fojtová, M and Houben, A and Puchta, H},
title = {CRISPR-Cas-mediated heritable chromosome fusions in Arabidopsis.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6775},
pages = {843-848},
doi = {10.1126/science.adz8505},
pmid = {41264695},
issn = {1095-9203},
mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Karyotype ; Meiosis ; Recombination, Genetic ; Gene Fusion ; },
abstract = {The genome of Arabidopsis thaliana consists of 10 chromosomes. By inducing CRISPR-Cas-mediated breaks at subcentromeric and subtelomeric sequences, we fused entire chromosome arms, obtaining two eight-chromosome lines. In one line, both arms of chromosome 3 were fused to chromosome 1. In another line, the arms were transferred to chromosomes 1 and 5. Both chromosome number-reduced lines were fertile. Phenotypic and transcriptional analyses revealed no differences compared with wild-type plants. After crossing with the wild type, the progeny showed reduced fertility. The meiotic recombination patterns of the transferred chromosome arms were substantially changed. Directed chromosome number changes in plants may enable new breeding strategies, redefining linkage groups and establishing genetic barriers. Moreover, our data indicate that plants are highly robust to engineered karyotype changes.},
}

*Arabidopsis/genetics
*Chromosomes, Plant/genetics
*CRISPR-Cas Systems
Gene Editing
*Genome, Plant
Karyotype
Meiosis
Recombination, Genetic
Gene Fusion

@article {pmid41263111,
year = {2025},
author = {Feng, X and Li, Y and Zheng, J and Chen, X and Yang, S and Chen, Y and Li, SC},
title = {MicrobialScope: an integrated genomic resource with rich annotations across bacteria, archaea, fungi, and viruses.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaf1234},
pmid = {41263111},
issn = {1362-4962},
support = {C2004-23Y//Young Collaborative Research/ ; JCYJ20220818101201004//Shenzhen Science and Technology Program/ ; 32300527//National Natural Science Foundation of China/ ; 32470695//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B0303040004//Key-Area Research and Development Program of Guangdong Province/ ; TC2024JC43//Basic Research Programs of Taicang, 2024/ ; //Shenzhen-Hong Kong Institute of Brain Science/ ; //SIAT-HKUST Joint Laboratory of Brain Science/ ; },
abstract = {Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth, playing critical roles in ecosystems, human health, and industrial applications. While existing microbial databases such as BV-BRC and IMG archive both monoisolate and metagenome-assembled genomes (MAGs) across domains, challenges remain in standardized, multi-level annotations and interactive tools for all microbial groups. Here, we present MicrobialScope (https://microbial.deepomics.org/), a comprehensive microbial genomic platform that integrates large-scale genome collections, multilevel annotations, and interactive visualizations. MicrobialScope harbors 2 411 503 bacterial, 24 472 archaeal, 20 203 fungal, and 188 267 viral genomes derived from both monoisolate assemblies and MAGs. Integrating 15 state-of-the-art bioinformatics tools and 10 specialized databases, MicrobialScope provides extensive annotations encompassing basic genomic features, genomic element prediction (e.g., genes, tRNAs, tmRNAs, CRISPR-Cas and anti-CRISPR elements, secondary metabolite biosynthetic clusters, signal peptides, and transmembrane proteins), and functional and structural annotations. This includes 1 072 114 935 proteins with diverse annotations, 24 640 186 tRNAs and tmRNAs, 140 888 CRISPR-Cas systems, 173 256 anti-CRISPR elements, 105 121 secondary metabolite biosynthetic clusters, 13 235 096 signal peptides, and 50 811 729 transmembrane proteins. In addition, MicrobialScope offers unrestricted access to all data resources, interactive visualization tools, and built-in online analytical modules for intuitive exploration and comparative analysis. With its extensive genome collection, comprehensive annotations, and user-friendly interface, MicrobialScope serves as a scalable platform to advance genome research across diverse microbial domains.},
}

@article {pmid41262458,
year = {2025},
author = {Gao, Z and Gao, Y and Wang, S and Li, X and Cao, W and Deng, W and Yao, L and Wei, X and Zhang, Z and Wang, S and Zhang, Y and Li, M and Xie, Y},
title = {Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases.},
journal = {Biosafety and health},
volume = {7},
number = {5},
pages = {312-322},
pmid = {41262458},
issn = {2590-0536},
abstract = {Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015-2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as "living therapeutics," can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.},
}

@article {pmid41262327,
year = {2025},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.},
journal = {Molecular systems design & engineering},
volume = {},
number = {},
pages = {},
pmid = {41262327},
issn = {2058-9689},
abstract = {The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.},
}

@article {pmid41262251,
year = {2025},
author = {Stelcer, E and Wozniak, A and Magner, D and Zeyland, J},
title = {Genetically modified pigs with α1,3-galactosyltransferase knockout and beyond: a comprehensive review of xenotransplantation strategies.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1663246},
pmid = {41262251},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Galactosyltransferases/genetics/deficiency ; *Animals, Genetically Modified ; Swine ; *Graft Rejection/immunology/genetics/prevention & control ; Gene Knockout Techniques ; Humans ; Heterografts/immunology ; },
abstract = {Xenotransplantation holds promise to eliminate the shortage of organs intended for humans in need. Pigs constitute the most suitable organ xenograft donor due to the fact that their organ anatomy physiological metabolism and immune system resemble those of humans. However, swine organs rapidly cause hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) after transplantation. HAR and AHXR are caused by the presence of xenoreactive natural immunoglobulins directed toward a galactose alpha1-3-galactose (alpha-Gal) epitope on porcine vascular endothelium. In order to suppress both types of rejection, pigs with alpha1,3-galactosyltransferase gene knockout (GT-KO) and other genetic modifications (like simultaneous expression of the human complementary regulatory proteins) are intensively investigated. This review highlights the usefulness of GT-KO pig - derived organs such as kidney, heart, corneal, and lung in xenotransplantation. To obtain transgenic pigs researchers can use several techniques based on pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT), viral transduction of DNA and DNA transposable element -based technology, site specific nucleases and modifications of the CRISPR/Cas bacterial immune system. Some additional strategies like targeted immunosuppression or tolerance induction of B and T cells will be essential for sustained survival of xenografts. Although xenotransplantation with the use of pigs is a very rapidly evolving field, more research is needed to create perfectly compatible with the human immune system organs.},
}

Animals
*Transplantation, Heterologous/methods
*Galactosyltransferases/genetics/deficiency
*Animals, Genetically Modified
Swine
*Graft Rejection/immunology/genetics/prevention & control
Gene Knockout Techniques
Humans
Heterografts/immunology

@article {pmid41261856,
year = {2025},
author = {Xiao, Y and Zhao, R and Bao, Y and Lu, B and Jiang, Y and Tang, Y and Li, B},
title = {Cas12a-assisted split crRNA complex for analysis and detection of diverse entities.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41261856},
issn = {1362-4962},
support = {SKL202402017//The Science and Technology Development Plan Project of Jilin Province/ ; SKL202302030//The Science and Technology Development Plan Project of Jilin Province/ ; 20240101005JJ//Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; 20230203193SF//Key R&D Program of Jilin Province/ ; 22525405//National Nature Science Foundation of China/ ; 22504138//National Nature Science Foundation of China/ ; 22474135//National Nature Science Foundation of China/ ; 22374142//National Nature Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Cas Systems ; MicroRNAs/genetics/analysis ; DNA, Single-Stranded/genetics ; DNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/methods ; },
abstract = {The Cas12a-crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a's application beyond nucleic acids. Using microRNA as a model, we validated the system's sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.},
}

*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Cas Systems
MicroRNAs/genetics/analysis
DNA, Single-Stranded/genetics
DNA/genetics
*Bacterial Proteins/genetics/metabolism
*Biosensing Techniques/methods

@article {pmid41261174,
year = {2025},
author = {Seker-Polat, F and Rogozinska, M and Ban, Y and Abdula, F and Buyukcelebi, K and Xie, P and Fan, J and Abbaszadeh, N and Kingham, Y and Paylakhi, SZ and Zhang, B and Adli, M},
title = {Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1610},
pmid = {41261174},
issn = {2399-3642},
support = {R01CA267544//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*B7-H1 Antigen/genetics/metabolism ; Humans ; *Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; *NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Animals ; Female ; Mice ; CRISPR-Cas Systems ; Pancreatic Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Ovarian Neoplasms/genetics ; },
abstract = {Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.},
}

*B7-H1 Antigen/genetics/metabolism
Humans
*Kelch-Like ECH-Associated Protein 1/metabolism/genetics
*NF-E2-Related Factor 2/metabolism/genetics
Cell Line, Tumor
*Gene Expression Regulation, Neoplastic
Animals
Female
Mice
CRISPR-Cas Systems
Pancreatic Neoplasms/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Signal Transduction
Ovarian Neoplasms/genetics

@article {pmid41260544,
year = {2025},
author = {Gupta, A and Beg, MA and Badhwar, S and Srivastava, S and Srivastava, R and Puri, N and Saxena, A and Abdin, MZ and Selvapandiyan, A},
title = {Nucleoside diphosphate kinase (LdNDK2): A metacyclogenesis-regulating kinase essential for Leishmania parasite survival within eukaryotic host cells.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108192},
doi = {10.1016/j.micpath.2025.108192},
pmid = {41260544},
issn = {1096-1208},
abstract = {Nucleoside diphosphate kinase (NDK) transfers phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism, benefiting both prokaryotes and eukaryotes. In Leishmania donovani, we identified a putative NDK2 (LdNDK2), hypothesized to play a crucial role in nucleotide metabolism and cellular energy regulation. To investigate its function and enzymatic properties, we cloned, expressed, and purified recombinant LdNDK2, confirming enzymatic activity via the ADP-Glo assay. The secondary structure and thermal stability were analyzed using circular dichroism spectroscopy, while intrinsic tryptophan fluorescence assays revealed that ATP's gamma phosphate is first transferred to rLdNDK2, forming an intermediate phospho-enzyme complex. We further evaluated the role of rLdNDK2 in ATP-mediated cytolysis of LPS-activated THP-1 cells by measuring lactate dehydrogenase release. Using the CRISPR-Cas9 method, we fluorescently tagged LdNDK2, localizing it to distinct regions of the endomembrane system. In vitro growth studies of LdNDK2 deleted (LdNDK2[-/-]) procyclic promastigotes, through CRISPR-Cas9, revealed that these mutants exhibit a longer flagellum, a reduced cell body, an altered cell division cycle, and increased growth compared to wild-type parasites. LdNDK2[-/-] parasites were arrested at the metacyclic stage, contrasting with continuous differentiation in parental counterparts. Morphological alterations and growth defects were confirmed by re-expressing LdNDK2 in knockouts. Infection with LdNDK2[-/-] parasites significantly reduced the viability and recovery of mast cells and THP-1 macrophages and flow cytometry indicated increased apoptosis in host cells, likely due to parasites inability to utilize NDK enzyme for preventing cytolysis. Our findings highlight LdNDK2's crucial role in metacyclogenesis and immune evasion, underscoring its therapeutic potential for leishmaniasis.},
}

@article {pmid41205603,
year = {2025},
author = {Regan, SB and Medhi, D and Xu, Y and White, TB and Jiang, YZ and Kim, JE and Wang, SC and Deng, Q and Jia, S and Baasan, D and Connelly, JP and Chang, TC and Pruett-Miller, SM and Jasin, M},
title = {Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4119-4137.e10},
doi = {10.1016/j.molcel.2025.10.015},
pmid = {41205603},
issn = {1097-4164},
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; Animals ; *Loss of Heterozygosity ; Mice ; DNA End-Joining Repair ; Gene Editing/methods ; Flow Cytometry ; CRISPR-Associated Protein 9/genetics/metabolism ; Epithelial Cells/metabolism ; },
abstract = {Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.},
}

*DNA Breaks, Double-Stranded
Humans
*CRISPR-Cas Systems
Animals
*Loss of Heterozygosity
Mice
DNA End-Joining Repair
Gene Editing/methods
Flow Cytometry
CRISPR-Associated Protein 9/genetics/metabolism
Epithelial Cells/metabolism

@article {pmid41194386,
year = {2025},
author = {Sanchez-Quirante, T and Kužmová, E and Riopedre-Fernandez, M and Golojuch, S and Vopálenský, P and Raindlová, V and El-Sagheer, AH and Brown, T and Hocek, M},
title = {Enzymatic Synthesis of Modified RNA Containing 5-Methyl- or 5-Ethylpyrimidines or Substituted 7-Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2755-2767},
doi = {10.1021/acschembio.5c00692},
pmid = {41194386},
issn = {1554-8937},
mesh = {*CRISPR-Cas Systems ; *Pyrimidines/chemistry/metabolism ; *RNA/chemistry/metabolism ; Humans ; *Purines/chemistry/metabolism ; Protein Biosynthesis ; DNA-Directed RNA Polymerases/metabolism ; Viral Proteins ; },
abstract = {A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.},
}

*CRISPR-Cas Systems
*Pyrimidines/chemistry/metabolism
*RNA/chemistry/metabolism
Humans
*Purines/chemistry/metabolism
Protein Biosynthesis
DNA-Directed RNA Polymerases/metabolism
Viral Proteins

@article {pmid41131871,
year = {2025},
author = {Campbell, KB and Ouye, RB and Wong, BL and Jiang, A and Okada, K and McKenney, RJ and Fisher, AJ and Beal, PA},
title = {Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2637-2648},
doi = {10.1021/acschembio.5c00493},
pmid = {41131871},
issn = {1554-8937},
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; *RNA-Binding Proteins/metabolism/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; RNA, Double-Stranded/metabolism/chemistry ; Protein Multimerization ; Binding Sites ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2'-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2'-OH at specific positions on the gRNA with a 2'-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2'-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2'-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2'-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2'-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2'-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.},
}

*Adenosine Deaminase/metabolism/chemistry/genetics
*RNA Editing
*RNA-Binding Proteins/metabolism/chemistry/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics
RNA, Double-Stranded/metabolism/chemistry
Protein Multimerization
Binding Sites

@article {pmid41099664,
year = {2025},
author = {Spavieri, JM and Inacio, TG and Seguchi, G and de Souza, BC and Pereira, GAG and de Mello, F},
title = {Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4412-4423},
doi = {10.1021/acssynbio.5c00316},
pmid = {41099664},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation/genetics ; Ethanol/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Glycerol/metabolism ; *CRISPR-Cas Systems/genetics ; *Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in Saccharomyces cerevisiae. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for GPD gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of GPD1 resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with GPD2 deletion, revealed the higher SEP among all tested strains. Furthermore, a GPD1-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.},
}

*Saccharomyces cerevisiae/genetics/metabolism
Fermentation/genetics
Ethanol/metabolism
*Saccharomyces cerevisiae Proteins/genetics/metabolism
Glycerol/metabolism
*CRISPR-Cas Systems/genetics
*Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism
Gene Editing/methods

@article {pmid41072420,
year = {2025},
author = {Zhao, G and Liu, Y and Zhang, G and Wang, J and Zou, Z and Wang, Y and Xu, S and Han, D and Xu, Z and Chen, Z and Yang, M and Zeng, Z and Wu, Y and Zhou, X and Huang, J},
title = {Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4138-4151.e4},
doi = {10.1016/j.molcel.2025.09.031},
pmid = {41072420},
issn = {1097-4164},
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; Animals ; Dependovirus/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; Mice ; },
abstract = {As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.},
}

Humans
*Gene Editing/methods
HEK293 Cells
Animals
Dependovirus/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genome, Human
CRISPR-Cas Systems
Mice

@article {pmid41017740,
year = {2025},
author = {Cao, X and Zeng, Z and Cao, X and He, Y and Wang, L and Li, D and Zhang, X},
title = {Generation and Phenotypic Analysis of the IL-10RAR104W/R104W Mouse Model.},
journal = {Inflammatory bowel diseases},
volume = {31},
number = {11},
pages = {3149-3159},
doi = {10.1093/ibd/izaf099},
pmid = {41017740},
issn = {1536-4844},
support = {32025023//National Natural Science Foundation of China/ ; 22YF1437700//Shanghai Science and Technology Program/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Phenotype ; *Inflammatory Bowel Diseases/genetics/pathology ; *Interleukin-10 Receptor alpha Subunit/genetics ; *Colitis/pathology/genetics ; *Point Mutation ; Humans ; Macrophages ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {BACKGROUND: Very-early-onset inflammatory bowel disease (VEO-IBD) is a form of IBD that manifests in infants and young children, with a significant proportion of them carrying interleukin 10 receptor alpha (IL-10RA) mutations. Despite the increasing incidence rate, the pathogenesis of VEO-IBD remains elusive, and treatment options are limited. The utilization of a humanized mouse model holds promise for further investigation into VEO-IBD. Previous study has revealed that VEO-IBD patients had a homozygous C > T mutation at IL-10RA position 301, which can be pathogenic.

METHODS: We generated the corresponding point mutation mouse model via CRISPR/Cas9 technology. Subsequently, we performed various experiments to assess the colitis phenotype in mice and conducted a preliminary exploration of the model's utility.

RESULTS: The mouse model progressively developed spontaneous colitis between 6 and 12 weeks. Hematoxylin and eosin (H&E) staining revealed abnormal colonic structure and massive local immune cell infiltration. The mouse model has abnormal levels of inflammatory cytokines in the colonic tissue, with an expansion of F4/80+ macrophages, CD4+ T cells, and B220+ B cells. Among the macrophages, the level of tissue-resident macrophages associated with anti-inflammation was reduced in IL-10RAR104W/R104W mice, while the level of immature macrophages associated with pro-inflammation was increased. Furthermore, we found that bone marrow transplantation can alter the composition of intestinal macrophage populations and treat intestinal inflammation in mutant mice. Finally, the result of subcutaneous tumor-bearing experiments indicated a faster tumor growth rate in the mutant mice.

CONCLUSIONS: In summary, we have successfully constructed a humanized mouse model with a stable spontaneous colitis phenotype, which is a valuable model for the therapeutic exploration of VEO-IBD.},
}

Animals
Mice
*Disease Models, Animal
Phenotype
*Inflammatory Bowel Diseases/genetics/pathology
*Interleukin-10 Receptor alpha Subunit/genetics
*Colitis/pathology/genetics
*Point Mutation
Humans
Macrophages
CRISPR-Cas Systems
Mice, Inbred C57BL

@article {pmid40954215,
year = {2025},
author = {Romero-Moya, D and Torralba-Sales, E and Calvo, C and Marin-Bejar, O and Magallon-Mosella, M and Distefano, M and Pera, J and Castaño, J and De Giorgio, F and Gonzalez, J and Iglesias, A and Berenguer-Balaguer, C and Schilling, M and Plass, M and Pasquali, L and Català, A and Molina, O and Wlodarski, MW and Bigas, A and Giorgetti, A},
title = {CRISPR-engineered human GATA2 deficiency model uncovers mitotic dysfunction and premature aging in HSPCs, impairing hematopoietic fitness.},
journal = {Leukemia},
volume = {39},
number = {12},
pages = {3015-3025},
pmid = {40954215},
issn = {1476-5551},
support = {PID2020-15591RB-100//Ministry of Economy and Competitiveness | Agencia Estatal de Investigación (Spanish Agencia Estatal de Investigación)/ ; FORT23/00032//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00014//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; KOG-202109-01162//European Hematology Association (EHA)/ ; 101029927//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; LCF-PR-HR24-00150//"la Caixa" Foundation (Caixa Foundation)/ ; PID2023-151556OB-I00//"la Caixa" Foundation (Caixa Foundation)/ ; PID2022-142966OB-I00//Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; *GATA2 Transcription Factor/genetics/deficiency ; *CRISPR-Cas Systems ; *Mitosis/genetics ; *GATA2 Deficiency/genetics/pathology ; Animals ; Mutation ; Mice ; Cell Proliferation ; *Cellular Senescence/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {GATA2 deficiency is a monogenic transcriptopathy disorder characterized by bone marrow failure (BMF), immunodeficiency, and a high risk of developing myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Although informative mouse models have been developed, the mechanisms by which GATA2 haploinsufficiency drives disease initiation in humans remain incompletely understood. To address this, we developed a novel humanized model using CRISPR/Cas9 technology to knock-in GATA2-R398W variant in primary cord blood CD34[+] cells. Additionally, we introduced specific mutations in SETBP1 and ASXL1 to model distinct premalignant stages of GATA2 deficiency. Through clonal competition and serial transplantation assays, we demonstrated that human CD34[+] cells harboring the GATA2 mutation exhibit significantly reduced fitness in vivo when compete with wild-type cells. Notably, this fitness disadvantage persists even when GATA2 mutations are combined with oncogenic SETBP1 and ASXL1 drivers, underscoring the dominant, deleterious effect of GATA2 deficiency on hematopoietic stem cell function. Functional in vitro analyses revealed that GATA2-R398W mutation impairs cell proliferation, disrupts cell cycle progression, and induces mitotic defects, which may contribute to hematopoietic stem/progenitor cell loss and impaired self-renewal. Transcriptomic profiles of GATA2-mutant cells revealed that these functional defects are associated with reduced HSC self-renewal capacity and upregulation of the pre-aging phenotype. Our work highlights the feasibility of generating a human GATA2 deficiency model suitable for studying the biological consequences of various GATA2 variants and the generation of a platform to test potential phenotype-rescuing therapeutics.},
}

Humans
*Hematopoietic Stem Cells/metabolism/pathology
*GATA2 Transcription Factor/genetics/deficiency
*CRISPR-Cas Systems
*Mitosis/genetics
*GATA2 Deficiency/genetics/pathology
Animals
Mutation
Mice
Cell Proliferation
*Cellular Senescence/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40753279,
year = {2025},
author = {Tan, GZH and Maurya, KS and Krishnamoorthi, S and Boonyaves, K and Urano, D},
title = {Regulatory helix deletion in glutamate decarboxylase reduces GABA and enhances Agrobacterium-mediated transient expression in lettuce.},
journal = {Journal of plant research},
volume = {138},
number = {6},
pages = {1033-1044},
pmid = {40753279},
issn = {1618-0860},
support = {A19E4a0101//Agency for Science, Technology and Research/ ; },
mesh = {*gamma-Aminobutyric Acid/metabolism ; *Glutamate Decarboxylase/genetics/metabolism ; *Lactuca/genetics/metabolism/enzymology ; *Agrobacterium/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Plant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.},
}

*gamma-Aminobutyric Acid/metabolism
*Glutamate Decarboxylase/genetics/metabolism
*Lactuca/genetics/metabolism/enzymology
*Agrobacterium/genetics
Gene Expression Regulation, Plant
Plants, Genetically Modified
*Plant Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41259334,
year = {2025},
author = {Siroosi, M and Ghasemi, F and Jabalameli, F and Emaneini, M and Salehi, M and Beigverdi, R and Amoozegar, MA},
title = {Investigating the impact of type I-E CRISPR-Cas systems and acrEI10 on multidrug-resistance in clinical isolates of Klebsiella pneumoniae.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335756},
doi = {10.1371/journal.pone.0335756},
pmid = {41259334},
issn = {1932-6203},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Klebsiella Infections/microbiology/drug therapy ; Microbial Sensitivity Tests ; beta-Lactamases/genetics ; },
abstract = {Klebsiella pneumoniae is a pathogen related to nosocomial infections with a high rate of antibiotic resistance. The aim of this study was to understand the impact of the presence of CRISPR-Cas systems and an anti-CRISPR gene on multidrug-resistance in K. pneumoniae isolates. The study analyzed 100 clinical K. pneumoniae isolates obtained from a hospital setting. The investigation involved determining antibiotic resistance profiles, including ESBL production, identifying specific carbapenemase and aminoglycoside resistance genes, detecting the presence of CRISPR-Cas systems, identifying the anti-CRISPR gene acrEI10, and sequencing CRISPR arrays. Correlation analysis between resistance genes and CRISPR-Cas systems was also performed. All isolates in this study were determined to be multidrug-resistant (MDR), with resistance rates exceeding 70% for the majority of antibiotics tested. The most prevalent carbapenemase genes were blaOXA-48 and blaNDM, while aminoglycoside resistance was primarily mediated by aac(6´)-Ia and ant(2")-Ia. Only 7% of the isolates harbored CRISPR-Cas systems and the gene acrEI10, which encodes an anti-CRISPR protein, was detected in one of the CRISPR-Cas positive isolates. Sequencing of the CRISPR array from this isolate showed similarities between the spacers and sequences found in plasmids and K. pneumoniae chromosome. No strong correlation was identified between the antibiotic resistance genes and CRISPR-Cas systems. Findings from this study suggest a complex interplay between these factors in MDR isolates of K. pneumoniae and show that further investigations are needed to have a better understanding of the mechanisms related to the coexistence of these elements and their impact on dissemination of antibiotic resistance genes.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification
*CRISPR-Cas Systems/genetics
*Drug Resistance, Multiple, Bacterial/genetics
Humans
*Bacterial Proteins/genetics
Anti-Bacterial Agents/pharmacology
Klebsiella Infections/microbiology/drug therapy
Microbial Sensitivity Tests
beta-Lactamases/genetics

@article {pmid41257956,
year = {2025},
author = {Feng, C and Wang, Y and Liu, C and Dong, F and Guo, M and Liu, F and Li, Y and Zhang, L},
title = {Programmable no-nonspecific genetic analytical system via dual-circle-based rolling circle amplification with an efficient CRISPR/Cas12a biosensing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40723},
pmid = {41257956},
issn = {2045-2322},
support = {CSTB2024NSCQ-MSX0785//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2023YQB061//Young Ph.D. Talent Incubation Program of the Second Affiliated Hospital of Army Medical University/ ; CQYC20220303658//Chongqing Science and Technology Innovation Leading Talent Support Program/ ; 2024ZDXM012//Chongqing Municipal Science and Health Joint Medical Research Project Key Project/ ; 82472384//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; DNA, Single-Stranded/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Circular/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
DNA, Single-Stranded/genetics
*Endodeoxyribonucleases/metabolism/genetics
DNA, Circular/genetics
Limit of Detection
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41256794,
year = {2025},
author = {Zhou, X and Ye, C and Xie, M and Wei, Y and Zhao, Y and Liu, X and Ma, J and Qing, J and Chen, Z},
title = {Advances in the application of CRISPR technology in pathogen detection: amplification-based and amplification-free strategies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1645699},
pmid = {41256794},
issn = {2235-2988},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/isolation & purification ; *Communicable Diseases/diagnosis ; },
abstract = {CRISPR technology, with its high specificity and programmability, has become an important tool for the detection of human pathogens. The timely and accurate detection of pathogens is crucial for public health. In recent years, significant progress has been made in the application of CRISPR technology for pathogen detection. However, several challenges remain, including detection sensitivity, specificity, and operational convenience. This review summarizes the latest advances in CRISPR technology for pathogen detection, with a focus on the principles and performance comparisons of amplification-based CRISPR (such as those combined with isothermal amplification techniques like RPA and LAMP) and amplification-free CRISPR (such as cascade CRISPR, sensor technologies, and digital droplet CRISPR). It also discusses their applications in pathogen detection. In addition, the article analyzes the advantages and limitations of CRISPR detection technology and looks forward to future development trends, providing a theoretical basis for the optimization of rapid diagnostic techniques for pathogens.},
}

Humans
*Molecular Diagnostic Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Sensitivity and Specificity
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/genetics/isolation & purification
*Communicable Diseases/diagnosis

@article {pmid41255389,
year = {2025},
author = {Pizzoccheri, R and Falchi, FA and Alloni, A and Caldarulo, M and Camboni, T and Zambelli, F and Pavesi, G and Visentin, C and Camilloni, C and Sertic, S and Briani, F},
title = {Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models.},
journal = {NAR molecular medicine},
volume = {2},
number = {1},
pages = {ugae028},
pmid = {41255389},
issn = {2976-856X},
abstract = {Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.},
}

@article {pmid41254867,
year = {2025},
author = {Podkowik, M and Tillman, A and Takats, C and Carion, H and Putzel, G and McWilliams, J and See, B and Wang, G and Munoz-Gomez, S and Otto, C and Drlica, K and Marraffini, L and Pironti, A and Hochman, S and Kerantzas, C and Shopsin, B},
title = {CRISPR-Cas-associated SCCmec variants in methicillin-resistant Staphylococcus aureus evade rapid diagnostic detection.},
journal = {The Journal of infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1093/infdis/jiaf575},
pmid = {41254867},
issn = {1537-6613},
abstract = {Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.},
}

@article {pmid41254256,
year = {2025},
author = {Wang, Q and Wang, Z and Liu, H and Lv, Y and Zhou, C and Li, C and Fan, H and Ouyang, X and Tao, L and Pei, X and Xie, T},
title = {PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1601},
pmid = {41254256},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
HEK293 Cells
*CRISPR-Associated Protein 9/metabolism/genetics
Animals
DNA Breaks, Double-Stranded

@article {pmid41253931,
year = {2025},
author = {Ni, J and Gong, J and Ran, Y and Bai, R and Huang, P and Zheng, Z and Zhou, M and Lan, F and Gu, W and Liu, X},
title = {Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40461},
pmid = {41253931},
issn = {2045-2322},
support = {JCYJ20220531091615034//Shenzhen Fundamental Research Program/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; INDEL Mutation ; },
abstract = {CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Gene Editing/methods
*Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Associated Protein 9/genetics/metabolism
Cell Line
INDEL Mutation

@article {pmid41252463,
year = {2025},
author = {Fehrenbach, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Baumdicker, F},
title = {An evolutionary approach to predict the orientation of CRISPR arrays.},
journal = {PLoS computational biology},
volume = {21},
number = {11},
pages = {e1013706},
doi = {10.1371/journal.pcbi.1013706},
pmid = {41252463},
issn = {1553-7358},
abstract = {CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.},
}

@article {pmid41252186,
year = {2025},
author = {Calhoun, CCS and Capps, MES and Muya, K and Gannaway, WC and Martina, V and Conklin, CL and Klein, MC and Webster, JM and Torija-Olson, EG and Thyme, SB},
title = {Removal of developmentally regulated microexons has a minimal impact on larval zebrafish brain morphology and function.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {41252186},
issn = {2050-084X},
mesh = {Animals ; *Zebrafish/genetics/growth & development ; *Brain/anatomy & histology/growth & development/physiology ; Larva/growth & development/genetics ; *Exons/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; *Alternative Splicing ; Phenotype ; },
abstract = {Microexon splicing is a vertebrate-conserved process through which small, often in-frame, exons are differentially included during brain development and across neuron types. Although the protein sequences encoded by these exons are highly conserved and can mediate interactions, the neurobiological functions of only a small number have been characterized. To establish a more generalized understanding of their roles in brain development, we used CRISPR/Cas9 to remove 45 microexons in zebrafish and assessed larval brain activity, morphology, and behavior. Most mutants had minimal or no phenotypes at this developmental stage. Among previously studied microexons, we uncovered baseline and stimulus-driven phenotypes for two microexons (meA and meB) in ptprd and reduced activity in the telencephalon in the tenm3 B0 isoform. Although mild neural phenotypes were discovered for several microexons that have not been previously characterized, including in ppp6r3, sptan1, dop1a, rapgef2, dctn4, vti1a, and meaf6. This study establishes a general approach for investigating conserved alternative splicing events and prioritizes microexons for downstream analysis.},
}

Animals
*Zebrafish/genetics/growth & development
*Brain/anatomy & histology/growth & development/physiology
Larva/growth & development/genetics
*Exons/genetics
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems
*Gene Expression Regulation, Developmental
*Alternative Splicing
Phenotype

@article {pmid41232532,
year = {2025},
author = {Yang, Y},
title = {What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.},
journal = {Neuron},
volume = {113},
number = {22},
pages = {3697-3702},
doi = {10.1016/j.neuron.2025.10.023},
pmid = {41232532},
issn = {1097-4199},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Brain Diseases/genetics/therapy ; *Precision Medicine/methods ; Infant ; },
abstract = {The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,[1] ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.},
}

Humans
*Gene Editing/methods
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
*Brain Diseases/genetics/therapy
*Precision Medicine/methods
Infant

@article {pmid41222482,
year = {2025},
author = {Chen, W and He, Y and Yuan, J and Yang, F},
title = {Catalytic hairpin assembly cascade-initiated proximity with self-priming amplification for CRISPR-enhanced ultrasensitive detection of coronary heart disease-associated microRNAs.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {45},
pages = {9254-9261},
doi = {10.1039/d5ay01456e},
pmid = {41222482},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Coronary Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; },
abstract = {Accurate detection of specific microRNAs (miRNAs) is essential for the early diagnosis of coronary heart disease. Emerging technologies, including functional nuclease-mediated target amplification and DNA nanotechnology, offer substantial potential for precise miRNA identification in clinical diagnostics. This study presents a highly sensitive and specific biosensing platform that integrates catalytic hairpin assembly (CHA) cascade-initiated proximity based self-priming amplification and CRISPR/Cas12a-mediated signal generation for miRNA quantification. Target miRNA initiates the CHA cascade, yielding a toehold-bearing CHA product. This toehold subsequently enables "Variable primer" extension, transcribing double-stranded DNA (dsDNA). The resultant dsDNA activates CRISPR/Cas12a, triggering collateral cleavage and signal amplification. Leveraging this dual-amplification strategy (CHA and CRISPR/Cas12a), the assay achieves a sub-femtomolar detection limit (0.36 fM). Dual-sequence verification, including CHA and CRISPR/Cas12a recognition, ensures exceptional specificity. Validation using spiked serum samples confirmed precise miRNA quantification. Collectively, this biosensor demonstrates significant promise for clinical molecular diagnostics.},
}

*MicroRNAs/genetics/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
*Coronary Disease/genetics/diagnosis
*Biosensing Techniques/methods

@article {pmid41217062,
year = {2025},
author = {Vieyra, F and Pindi, C and Lisi, GP and Morzan, UN and Palermo, G},
title = {Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants.},
journal = {The journal of physical chemistry. B},
volume = {129},
number = {46},
pages = {11949-11958},
doi = {10.1021/acs.jpcb.5c06153},
pmid = {41217062},
issn = {1520-5207},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Gene Editing ; },
abstract = {Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.},
}

Molecular Dynamics Simulation
*CRISPR-Cas Systems
DNA/chemistry/metabolism/genetics
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
Gene Editing

@article {pmid41183414,
year = {2026},
author = {Luo, Q and Zheng, C and Huang, Y and Liang, L and Gong, Y and Zhang, J and Tang, Q and Zhang, K and Liao, X},
title = {Polydopamine-stabilized CsPbBr3 enables toxicity-reduced ECL detection of MMP-2 via CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118193},
doi = {10.1016/j.bios.2025.118193},
pmid = {41183414},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Indoles/chemistry ; *Polymers/chemistry ; *Matrix Metalloproteinase 2/isolation & purification/chemistry/blood ; Gold/chemistry ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Lead/chemistry ; Titanium/chemistry ; },
abstract = {Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb[2+] safety concerns. We construct a CsPbBr3@PDA-Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr3, suppressing trap-mediated quenching and mitigating Pb[2+] release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide-DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from "off" to "on." Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr3; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.},
}

*Biosensing Techniques/methods
*Indoles/chemistry
*Polymers/chemistry
*Matrix Metalloproteinase 2/isolation & purification/chemistry/blood
Gold/chemistry
Electrochemical Techniques/methods
CRISPR-Cas Systems
Humans
Limit of Detection
Luminescent Measurements/methods
Lead/chemistry
Titanium/chemistry

@article {pmid41183412,
year = {2026},
author = {Liu, Y and Li, H and Wang, J and Liu, J and Zhou, X},
title = {Dual-acting CRISPR/Cas12a system enhanced hydrogel fluorescent aptasensor for one-pot detection of tetracycline in water.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118181},
doi = {10.1016/j.bios.2025.118181},
pmid = {41183412},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Tetracycline/analysis/isolation & purification ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation & purification ; *Anti-Bacterial Agents/analysis ; Water/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid yet ultrasensitive and accurate detection of tetracycline (TC) in aquatic environments subjected to composite pollution remains challenging. CRISPR-mediated biosensors have been extensively studied to achieve ultrasensitive detection. However, these biosensors have always been coupled with a time-consuming nucleic acid amplification process to improve the sensitivity, which may cause erratic signal due to the sophisticated biochemical reaction cascades, hence leading to inaccurate results. To address this issue, we proposed a novel amplification-free fluorescent aptasensor based on polyacrylamide-deoxyribonucleic acid hydrogel integrated with a dual-acting CRISPR/Cas12a system for ultrasensitive and rapid detection of TC in water. With this well-designed dual-acting CRISPR/Cas12a system, a single aptamer-target specific molecular binding event can synchronously initiate pairing between two distinct CRISPR RNA and their target nucleic acid modified on the hydrogel, enabling dual activation of Cas12a. The aptasensor instantly emitted quantifiable strong fluorescence due to the efficient cleavage of reporter probes by the twofold activated Cas12a, demonstrating a TC detection limit of 0.035 μg/L, with approximately 10.6-fold and 5.6-fold sensitivity improvement over the two corresponding single-crRNA systems. The entire detection process can be accomplished in one pot within 10 min. The one-step hydrogel aptasensor shows superior resistance to matrix interference over the conventional solution-phase system, achieving satisfactory recovery percentages (92 %-105 %) for TC in different water matrices. This study offers a new perspective on CRISPR/Cas12a biosensor design and advances the environmental antibiotic monitoring field.},
}

*Biosensing Techniques/methods
*Tetracycline/analysis/isolation & purification
*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
Hydrogels/chemistry
Limit of Detection
*Water Pollutants, Chemical/analysis/isolation & purification
*Anti-Bacterial Agents/analysis
Water/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41175692,
year = {2026},
author = {Hao, X and Qin, C and He, C and Yuan, Q and Yang, P and Wen, Y and Wu, Y and Cui, R and Zhang, Y and Chen, W and Liu, Y and Ramadan, S and Ying, Y and Li, D and Xu, L},
title = {In situ detection of bacteria from skin interstitial fluid via CRISPR microneedles: An amplification-free platform for point-of-care diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118123},
doi = {10.1016/j.bios.2025.118123},
pmid = {41175692},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Staphylococcus aureus/isolation & purification/genetics ; Humans ; *Skin/microbiology ; *Extracellular Fluid/microbiology ; CRISPR-Cas Systems ; Gold/chemistry ; Graphite/chemistry ; Limit of Detection ; *DNA, Bacterial/genetics/isolation & purification ; Wearable Electronic Devices ; Needles ; Point-of-Care Systems ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {We report a CRISPR/Cas12a-functionalized microneedle (MN) biosensor for in situ, amplification-free detection of pathogenic bacteria directly from skin interstitial fluid. The platform integrates conductive poly(styrene)/gold/graphene oxide MNs with a minimized electrochemical signal transducer, enabling real-time and highly specific sensing of bacterial DNA through CRISPR-mediated cleavage of ferrocene-labeled ssDNA reporters. Using Staphylococcus aureus as a model target, this strategy achieves direct detection limits of 0.69 pM in ex situ assays and 6.3 pM in situ, without the need for target amplification. The sensor also detects bacterial loads as low as 4.27 × 10[5] CFU/mL in interstitial fluid, which is below typical clinical thresholds. Engineered for biocompatibility and minimal invasiveness, the MNs effectively access interstitial fluid, capture targets and produce quantifiable electrochemical signals within 1 h. Paired with a portable reader and smartphone interface, this wearable system offers a user-friendly tool for point-of-care diagnostics and personalized infectious disease monitoring.},
}

*Biosensing Techniques/instrumentation
*Staphylococcus aureus/isolation & purification/genetics
Humans
*Skin/microbiology
*Extracellular Fluid/microbiology
CRISPR-Cas Systems
Gold/chemistry
Graphite/chemistry
Limit of Detection
*DNA, Bacterial/genetics/isolation & purification
Wearable Electronic Devices
Needles
Point-of-Care Systems
*Staphylococcal Infections/microbiology/diagnosis

@article {pmid41161233,
year = {2026},
author = {Shan, J and Wang, W and Sheng, Y and Hong, B and Luo, L and Liu, X and Ma, Y and Wang, J},
title = {Ultra-fast one-pot isothermal detection of respiratory virus: ADNA-initiated CRISPR/Cas12a-mediated RCA cycle.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118141},
doi = {10.1016/j.bios.2025.118141},
pmid = {41161233},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/isolation & purification/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; *COVID-19/diagnosis/virology ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*SARS-CoV-2/isolation & purification/genetics
*Biosensing Techniques/methods
Humans
Limit of Detection
*Molecular Diagnostic Techniques/methods
*COVID-19/diagnosis/virology
Endodeoxyribonucleases/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41138445,
year = {2026},
author = {Hu, M and Liu, F and Zhang, J and Yang, L and Yan, Y and Wang, Y and Liu, M and An, H and Zhu, L and Ai, Y and Jiang, X},
title = {Integrated CRISPR/Cas12a-activated liposomal SERS amplification in microfluidic chips for ultrasensitive ampicillin detection.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118131},
doi = {10.1016/j.bios.2025.118131},
pmid = {41138445},
issn = {1873-4235},
mesh = {*Ampicillin/analysis/isolation & purification ; Liposomes/chemistry ; CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Biosensing Techniques ; Limit of Detection ; *Anti-Bacterial Agents/analysis ; Animals ; Gold/chemistry ; Lab-On-A-Chip Devices ; Milk/chemistry ; Silver/chemistry ; Humans ; Metal Nanoparticles/chemistry ; },
abstract = {The misuse of antibiotics has created a vicious cycle: from excessive use in livestock breeding to elevated residue levels in food products, ultimately leading to heightened antibiotic resistance in humans, a phenomenon with significant public health consequences. Food-based antibiotic testing is crucial for preventing antibiotic misuse and protecting human health. However, traditional analytical methods exhibit high equipment dependency, restricting on-site detection in hills, plateaus, and other regions with inconvenient transportation. This study developed an integrated CRISPR/Cas12a-activated liposomal Surface-enhanced Raman scattering (SERS) amplification system within a microfluidic chip for ultrasensitive ampicillin detection. The method achieves primary signal amplification by converting ampicillin concentration into a DNA signal through catalytic hairpin assembly (CHA), followed by secondary signal amplification using an Au@Ag substrate coupled with SERS technology for 4-MPBA@liposome marker detection. The platform demonstrated a linear detection range of 1 fM-1 nM with a limit of detection as low as 740 aM. Notably, the method exhibited excellent stability, selectivity, and sensitivity, successfully detecting ampicillin residues in environmental water and milk samples. It provides a technological approach for environmental monitoring and food safety, demonstrating significant practical application value.},
}

*Ampicillin/analysis/isolation & purification
Liposomes/chemistry
CRISPR-Cas Systems/genetics
*Spectrum Analysis, Raman/methods
*Biosensing Techniques
Limit of Detection
*Anti-Bacterial Agents/analysis
Animals
Gold/chemistry
Lab-On-A-Chip Devices
Milk/chemistry
Silver/chemistry
Humans
Metal Nanoparticles/chemistry

@article {pmid41108536,
year = {2025},
author = {Siddiqui, S and Siddiqui, H and Riguene, E and Nomikos, M},
title = {Zebrafish: A Versatile and Powerful Model for Biomedical Research.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70080},
pmid = {41108536},
issn = {1521-1878},
support = {//NPRP-Blue Skies Research Award/ ; NPRP-BSRA1-0504-210082//Qatar Research Development and Innovation Council (QRDI), Doha, Qatar/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Humans ; *Biomedical Research/methods ; *Disease Models, Animal ; Precision Medicine/methods ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Zebrafish (Danio rerio) have become a versatile model in precision medicine, bridging fundamental biology with translational applications. Their optical transparency, rapid development, and high genetic conservation with humans enable real-time imaging and cost-efficient high-throughput screening. Advances in CRISPR/Cas9, prime editing, and morpholino approaches have expanded their utility for modeling diverse human diseases. In addition to well-established roles in cardiovascular, neurological, metabolic, oncological, and infectious disease research, emerging applications include non-invasive larval urine assays, functional validation of rare human variants, host-microbiome interactions, and automated behavioral profiling for neuropsychiatric conditions. Limitations such as species-specific lipid metabolism and limited antibody availability remain, yet recent integration of single-cell transcriptomics, computational modeling, and machine learning is enhancing translational relevance. Collectively, these innovations position zebrafish as a scalable and powerful platform for disease modeling and personalized therapeutic strategies, underscoring their growing impact in the evolving landscape of precision medicine.},
}

*Zebrafish/genetics
Animals
Humans
*Biomedical Research/methods
*Disease Models, Animal
Precision Medicine/methods
CRISPR-Cas Systems
Gene Editing

@article {pmid41101316,
year = {2025},
author = {Chitboonthavisuk, C and Martin, C and Huss, P and Peters, JM and Anantharaman, K and Raman, S},
title = {Systematic genome-wide mapping of host determinants of bacteriophage infectivity.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101427},
doi = {10.1016/j.cels.2025.101427},
pmid = {41101316},
issn = {2405-4720},
mesh = {*Escherichia coli/virology/genetics ; *Bacteriophages/genetics/pathogenicity ; *Host-Pathogen Interactions/genetics ; CRISPR-Cas Systems/genetics ; Genome, Viral/genetics ; Bacteriophage T7/genetics/pathogenicity ; Host Microbial Interactions/genetics ; },
abstract = {Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed phage-host analysis using genome-wide CRISPR interference and phage packaging ("PHAGEPACK"), a pooled assay that systematically and comprehensively measures each host gene's impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways that affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates that phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers insights into phage-host interactions, phage evolution, and bacterial resistance.},
}

*Escherichia coli/virology/genetics
*Bacteriophages/genetics/pathogenicity
*Host-Pathogen Interactions/genetics
CRISPR-Cas Systems/genetics
Genome, Viral/genetics
Bacteriophage T7/genetics/pathogenicity
Host Microbial Interactions/genetics

@article {pmid41101079,
year = {2026},
author = {Ma, Q and Rong, Z and Shen, J and Yang, M and Han, Y},
title = {Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117197},
doi = {10.1016/j.jpba.2025.117197},
pmid = {41101079},
issn = {1873-264X},
mesh = {Humans ; *Janus Kinase 2/genetics ; *CRISPR-Cas Systems/genetics ; *Myeloproliferative Disorders/genetics/diagnosis ; Mutation ; Sensitivity and Specificity ; Recombinases ; Real-Time Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.},
}

Humans
*Janus Kinase 2/genetics
*CRISPR-Cas Systems/genetics
*Myeloproliferative Disorders/genetics/diagnosis
Mutation
Sensitivity and Specificity
Recombinases
Real-Time Polymerase Chain Reaction/methods
Nucleic Acid Amplification Techniques/methods

@article {pmid41047505,
year = {2025},
author = {Rajalekshmi, S and Sathyan, KM},
title = {Direct Protein Degradation: Emerging Tools to Probe Biological Complexity in Mammalian Systems.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70075},
doi = {10.1002/bies.70075},
pmid = {41047505},
issn = {1521-1878},
support = {//School of Medicine, Southern Illinois University/ ; },
mesh = {Animals ; *Proteolysis ; Humans ; Gene Editing/methods ; Mammals/metabolism ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Conditional degron approaches for acute and reversible protein depletion have become standard tools for studying gene function in cells and model organisms. Traditional gene perturbation methods have advanced gene function studies but are limited by slow kinetics, potential irreversibility, and lethality when targeting essential genes. To overcome these limitations, tag-based and antibody-based direct protein degradation technologies have been developed. These direct protein degradation systems utilize endogenous protein degradation pathways to achieve rapid and reversible protein depletion. When combined with genome editing, these systems provide precise temporal-and in some cases, spatial-control over endogenous protein expression. In this review, we will discuss the current status of tag-based and antibody-based direct protein degron technologies. We aim to provide a comprehensive guide for selecting these tools, highlighting their context-dependent applications and potential improvements to enhance efficiency and reliability.},
}

Animals
*Proteolysis
Humans
Gene Editing/methods
Mammals/metabolism
*Proteins/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41045937,
year = {2025},
author = {Koo, BM and Todor, H and Sun, J and van Gestel, J and Hawkins, JS and Hearne, CC and Banta, AB and Huang, KC and Peters, JM and Gross, CA},
title = {Comprehensive genetic interaction analysis of the Bacillus subtilis envelope using double-CRISPRi.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101406},
doi = {10.1016/j.cels.2025.101406},
pmid = {41045937},
issn = {2405-4720},
mesh = {*Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Regulatory Networks/genetics ; *Cell Wall/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Peptidoglycan ; CRISPR-Cas Systems/genetics ; },
abstract = {Understanding bacterial gene function remains a major challenge. Double-mutant genetic interaction analysis addresses this challenge by uncovering the functional partners of targeted genes, enabling association of genes of unknown function with known pathways and unraveling of connections among well-studied pathways, but such approaches are difficult to implement at the genome scale. Here, we use double-CRISPR interference (CRISPRi) to systematically quantify genetic interactions at scale for the Bacillus subtilis cell envelope, including essential genes. We discover >1,000 genetic interactions, some known and others novel. Our analysis pipeline and experimental follow-ups reveal the shared and distinct roles of paralogous genes such as mreB and mbl in peptidoglycan and teichoic acid synthesis and identify additional genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse species. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Bacillus subtilis/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Gene Regulatory Networks/genetics
*Cell Wall/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Peptidoglycan
CRISPR-Cas Systems/genetics

@article {pmid41045936,
year = {2025},
author = {Dénéréaz, J and Eray, E and Jana, B and de Bakker, V and Todor, H and van Opijnen, T and Liu, X and Veening, JW},
title = {Dual CRISPRi-seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101408},
doi = {10.1016/j.cels.2025.101408},
pmid = {41045936},
issn = {2405-4720},
mesh = {*Streptococcus pneumoniae/genetics ; *Cell Cycle/genetics ; *CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Regulatory Networks/genetics ; Genome-Wide Association Study/methods ; },
abstract = {Uncovering genotype-phenotype relationships is hampered by genetic redundancy. For example, most genes in Streptococcus pneumoniae are non-essential under laboratory conditions. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. We developed a broadly applicable dual CRISPRi-seq method and analysis pipeline to probe genetic interactions (GIs) genome-wide. A library of 869 dual single-guide RNAs (sgRNAs) targeting high-confidence operons was created, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified. Besides known GIs, we found previously unknown positive and negative interactions involving genes in fundamental cellular processes such as division and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. All interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN), which can serve as a starting point for new biological discoveries. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*Streptococcus pneumoniae/genetics
*Cell Cycle/genetics
*CRISPR-Cas Systems/genetics
Genome, Bacterial/genetics
Computational Biology/methods
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Regulatory Networks/genetics
Genome-Wide Association Study/methods

@article {pmid40991130,
year = {2025},
author = {Kapoor, SA and Choudhary, P and Kasana, RC},
title = {Exploring CRISPR/Cas9-Mediated Gene Editing Advances in Conventional and Non-conventional Yeast Species.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {11},
pages = {7083-7122},
pmid = {40991130},
issn = {1559-0291},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Yeasts/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {In recent years, using modern technologies, researchers have harnessed the potential of yeast species for various industrial uses, such as the bioproduction of biopharmaceuticals, food additives, industrial biocatalysts, and biofuels. To improve the efficiency and potential of yeast species for industrial uses, genetic modification is carried out. Various genome engineering techniques, including Cre-loxP, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have been employed by different research groups for the genetic manipulation of yeast species. Among different genome engineering techniques, CRISPR/Cas9 has become popular because of its precise editing at targeted loci with increased efficiency. The ease of use, effectiveness, and adaptability of CRISPR/Cas9 make multiplexing possible for simultaneously targeting multiple genes, which was earlier very challenging through traditional methods. Moreover, the ability to perform marker-free editing is the significant advantage offered by CRISPR/Cas9. This review focuses on the applications of the CRISPR/Cas9 system in both conventional and non-conventional yeast species. Further, we discussed the advancements of CRISPR/Cas9, including the regulation of gene transcription-activation/repression and other genome engineering aspects. Additionally, innovations in CRISPR/Cas9, such as cloning-free CRISPR/Cas9 assembly, CRISPR-targeted in vivo editing (ACtive), CRISPR/Cas9-induced gene conversion, and selective ploidy ablation (CRI-SPA) are also discussed for enhancing the potential applications of CRISPR/Cas9 in diverse yeast species.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Yeasts/genetics
*Saccharomyces cerevisiae/genetics

@article {pmid41249581,
year = {2025},
author = {Turgeman-Grott, I and Golan, N and Neri, U and Naki, D and Altman-Price, N and Eizenshtein, K and Choudhary, DK and Levy, R and Navok, S and Cohen, L and Shalev, Y and Singla, H and Reshef, L and Gophna, U},
title = {A previously undescribed archaeal virus suppresses host immunity.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {41249581},
issn = {1469-3178},
support = {787514//EC | European Research Council (ERC)/ ; 1599/24//Israel Science Foundation (ISF)/ ; },
abstract = {Extremophilic archaea can have chronic viral infections that are well-tolerated by the hosts and may potentially protect against more lethal infections. Here we show that a natural Haloferax strain (48N), is chronically infected by a lemon-shaped virus. This viral infection is not cleared spontaneously, despite the multiple defense systems of the host. Curing 48N of its virus led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of ~107 min at 45 °C, and faster than any known Haloferax species at this temperature. The virus subverts host defenses by reducing its transcription, including the CRISPR spacer acquisition machinery. Nonetheless, even in the virus-cured background, spacer acquisition is very low, indicating that another genetic element is disrupting CRISPR activity. Our results suggest that the slow growth of some halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.},
}

@article {pmid41249545,
year = {2025},
author = {Warkad, S and Kumar, A and Gampa, M and Goswami, S and T, V and Kumar, S and Dalal, M and Mishra, D and Jha, GK and C, V and Kumar, RR},
title = {Small RNAs big impact: a review on microRNA-mediated tolerance in wheat under terminal heat.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {245},
pmid = {41249545},
issn = {1438-7948},
support = {CABin//Indian Council of Agricultural Research/ ; NICRA//Indian Council of Agricultural Research/ ; },
mesh = {*Triticum/genetics/physiology ; *MicroRNAs/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; *Heat-Shock Response/genetics ; *RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRNAs) are key regulators of gene expression in plant responses to abiotic stresses, including heat stress. High temperatures during the critical developmental stages of wheat (Triticum aestivum L.) drastically limit growth and production. Recent research has found that specific miRNAs regulate molecular complexes and physiological responses by targeting transcription factors, heat shock proteins, and signaling components, thereby modulating heat stress tolerance pathways. This review highlights current knowledge about heat-responsive miRNAs in wheat, including their validated targets and functional involvement in thermotolerance. In addition, we summarized the potential CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR associated protein 9) genome editing tool for precise alteration of miRNA genes or their targets, degradome profiling, the secondary structure of miRNA, and how interplay of miRNAs with HSFs and HSPs in target gene regulation to improve heat resilience. A comprehensive understanding of miRNA-regulated networks presents novel possibilities for developing climate-resilient wheat varieties, thereby ensuring food security in the face of global warming.},
}

*Triticum/genetics/physiology
*MicroRNAs/genetics/metabolism
Gene Expression Regulation, Plant
*Thermotolerance/genetics
*Heat-Shock Response/genetics
*RNA, Plant/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41249169,
year = {2025},
author = {Ramadoss, GN and Namaganda, SJ and Kumar, MM and Hamilton, JR and Sharma, R and Chow, KG and Workley, LA and Macklin, BL and Sun, M and Ha, AS and Liu, JC and Fellmann, C and Watry, HL and Dierks, PH and Bose, RS and Jin, J and Perez, BS and Sandoval Espinoza, CR and Matia, MP and Lu, SH and Judge, LM and Shy, BR and Nussenzweig, A and Adamson, B and Murthy, N and Doudna, JA and Kampmann, M and Conklin, BR},
title = {Characterizing and controlling CRISPR repair outcomes in nondividing human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9883},
pmid = {41249169},
issn = {2041-1723},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; *Neurons/metabolism/cytology ; Myocytes, Cardiac/metabolism ; DNA Damage ; T-Lymphocytes/metabolism ; },
abstract = {Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.},
}

Humans
Induced Pluripotent Stem Cells/metabolism/cytology
*Gene Editing/methods
*DNA Repair/genetics
*CRISPR-Cas Systems/genetics
*Neurons/metabolism/cytology
Myocytes, Cardiac/metabolism
DNA Damage
T-Lymphocytes/metabolism

@article {pmid41248011,
year = {2025},
author = {Dereki, I and Chondrou, V and Lagoumintzis, G and Sgourou, A},
title = {Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69328},
pmid = {41248011},
issn = {1940-087X},
mesh = {Humans ; *DNA Methylation/genetics ; K562 Cells ; DNA Methyltransferase 3A ; *DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis ; Plasmids/genetics ; *Histone Deacetylase 1/genetics/metabolism ; Genetic Vectors/genetics ; Recombinant Fusion Proteins/genetics/biosynthesis ; *Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
abstract = {Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.},
}

Humans
*DNA Methylation/genetics
K562 Cells
DNA Methyltransferase 3A
*DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis
Plasmids/genetics
*Histone Deacetylase 1/genetics/metabolism
Genetic Vectors/genetics
Recombinant Fusion Proteins/genetics/biosynthesis
*Epigenesis, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems

@article {pmid41243026,
year = {2025},
author = {Menelih, A and Girma, A and Assamere, A},
title = {Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {243},
pmid = {41243026},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Epigenome ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Plants/genetics ; DNA Methylation ; *Genome, Plant ; Epigenome Editing ; },
abstract = {Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.},
}

*Gene Editing/methods
*Epigenome
CRISPR-Cas Systems
*Epigenesis, Genetic
*Plants/genetics
DNA Methylation
*Genome, Plant
Epigenome Editing

@article {pmid41242874,
year = {2025},
author = {Akter, T and Baishnab, SM and Damtie, M and Dumpati, S and Eshete, TB and Irfan, S and Kumar, M and Sara, M and Urmi, UL and Willcox, M},
title = {A review of technological advances in the management of bacterial, fungal, viral, and Acanthamoeba keratitis.},
journal = {Clinical & experimental optometry},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/08164622.2025.2579169},
pmid = {41242874},
issn = {1444-0938},
abstract = {Microbial keratitis is a major cause of monocular blindness worldwide. Trauma and contact lens wear are major risk factors. Bacteria are a major cause, but fungi, viruses, and Acanthamoeba can also cause the disease. Therapeutic options are dwindling with the rise of antimicrobial resistance in all of these microbes, but especially bacteria. Infection with resistant strains results in worse clinical outcomes. New antimicrobial therapies are being developed but most have yet to be either used to treat ocular infections or become commercially available. For bacterial keratitis, antimicrobial peptides (AMPs) are a potential new option, as are bacteriophages and even bacteria that eat other bacteria or maggot secretions. For fungal keratitis, ocular natamycin, with oral ketoconazole or voriconazole treatments, offer improved outcomes for severe cases. For viral keratitis, AMPs again offer hope as new therapeutics, as do RNA interference and CRISPR-Cas systems. The recent release of a commercially available polyhexamethylene biguanide eye drop is a step forward, but new therapies that can kill both Acanthamoeba trophozoites and cysts are still needed. Beyond antibiotics, photoactivated chromophores for keratitis-corneal collagen cross-linking and photodynamic therapy have been successfully used. Therapies for large ulcers or scarring include corneal gluing, tissue scaffolds such as amniotic membrane or conjunctival flaps, and tissue replacement through penetrating or lamellar keratoplasty.},
}

@article {pmid41242543,
year = {2025},
author = {Hillary, VE and Rajan, V and Ceasar, SA},
title = {CRISPR's impact on cancer: From fundamental models to clinical solutions.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124087},
doi = {10.1016/j.lfs.2025.124087},
pmid = {41242543},
issn = {1879-0631},
abstract = {CRISPR is a programmable genome editing method that has transformed genome engineering and advanced cancer research. Various engineering iterations of CRISPR, including base and prime editing, facilitate more accurate and adaptable genomic manipulations, opening new frontiers in understanding and combating cancer. This review emphasizes CRISPR's significant influence in cancer biology, from its ability to accurately recreate somatic mutations in cellular and animal models to its application in developing precise diagnostic tools. We also examine advancements in cancer therapies, particularly through the ex vivo engineering of robust chimeric antigen receptor (CAR) T cells that perform effectively in restrictive tumor microenvironments. Lastly, we consider the existing limitations of CRISPR-Cas systems and outline future directions that could further enhance the application of this powerful technology against cancer.},
}

@article {pmid41213415,
year = {2026},
author = {Xu, YY and Zhou, SM and Wang, LY and Zhang, R and Li, K and Qian, ZY and Xiao, L},
title = {Methods for detecting off-target effects of CRISPR/Cas9.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108750},
doi = {10.1016/j.biotechadv.2025.108750},
pmid = {41213415},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.},
}

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

@article {pmid41208769,
year = {2025},
author = {Luo, D and Wang, N and Major-Monfried, H and Ralls, J and Rha, S and Maitland, SA and Ponnienselvan, K and Yamada, M and Bauer, DE and Wolfe, SA and Kentsis, A},
title = {Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {11},
pages = {2472-2486},
doi = {10.1021/acs.bioconjchem.5c00440},
pmid = {41208769},
issn = {1520-4812},
mesh = {Humans ; *Cell-Penetrating Peptides/chemistry ; *Drug Delivery Systems/methods ; *Macromolecular Substances/chemistry ; Gene Editing ; Cell Line, Tumor ; Peptidomimetics/chemistry/pharmacology ; Ligands ; CRISPR-Cas Systems ; },
abstract = {Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.},
}

Humans
*Cell-Penetrating Peptides/chemistry
*Drug Delivery Systems/methods
*Macromolecular Substances/chemistry
Gene Editing
Cell Line, Tumor
Peptidomimetics/chemistry/pharmacology
Ligands
CRISPR-Cas Systems

@article {pmid41186998,
year = {2025},
author = {Wang, W and Liu, H and Yang, Z and Wang, L and Li, W and He, H and Chen, S and Xu, M and Lu, Y},
title = {Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in Saccharopolyspora spinosa.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {46},
pages = {29717-29728},
doi = {10.1021/acs.jafc.5c08276},
pmid = {41186998},
issn = {1520-5118},
mesh = {*Saccharopolyspora/genetics/metabolism/growth & development ; *Macrolides/metabolism ; *Bacterial Proteins/metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Multigene Family ; Gene Expression Regulation, Bacterial ; *Insecticides/metabolism ; },
abstract = {Spinosyns, polyketide-derived macrolide insecticides produced by Saccharopolyspora spinosa, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (gltA1, fabH3, fabH4, and glgC) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (pyc or gltA2) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of pyc and gltA2, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in S. spinosa achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.},
}

*Saccharopolyspora/genetics/metabolism/growth & development
*Macrolides/metabolism
*Bacterial Proteins/metabolism/genetics
Metabolic Engineering/methods
CRISPR-Cas Systems
Multigene Family
Gene Expression Regulation, Bacterial
*Insecticides/metabolism

@article {pmid41170688,
year = {2025},
author = {Wang, S and Guan, X and Sun, S},
title = {A CRISPR/Cas12a-based aptasensor enhanced by functionalized AuNPs for sensitive full-range C-reactive protein detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {45},
pages = {14749-14760},
doi = {10.1039/d5tb01727k},
pmid = {41170688},
issn = {2050-7518},
mesh = {*C-Reactive Protein/analysis ; *Gold/chemistry ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Particle Size ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections and autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng mL[-1] and a quantifiable range of 0.1-150 μg mL[-1]. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated results that are in strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical applications.},
}

*C-Reactive Protein/analysis
*Gold/chemistry
*Aptamers, Nucleotide/chemistry/metabolism
Humans
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
Particle Size
*Endodeoxyribonucleases/metabolism
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41151122,
year = {2025},
author = {Cea Salazar, VI and Boender, AJ and Seelke, AMH and Gaard, L and Mederos, SL and Rogers, S and Gutierrez, XZ and Bales, KL and Young, LJ and Trainor, BC},
title = {CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice.},
journal = {Hormones and behavior},
volume = {176},
number = {},
pages = {105845},
doi = {10.1016/j.yhbeh.2025.105845},
pmid = {41151122},
issn = {1095-6867},
mesh = {Animals ; *Receptors, Oxytocin/genetics/metabolism ; Female ; *Stress, Psychological/genetics/metabolism ; Peromyscus ; Mice ; *Amygdala/metabolism ; *Social Behavior ; Gene Knockdown Techniques ; Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Septal Nuclei/metabolism ; *Avoidance Learning/physiology ; Behavior, Animal/physiology ; },
abstract = {Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific.},
}

Animals
*Receptors, Oxytocin/genetics/metabolism
Female
*Stress, Psychological/genetics/metabolism
Peromyscus
Mice
*Amygdala/metabolism
*Social Behavior
Gene Knockdown Techniques
Nucleus Accumbens/metabolism
CRISPR-Cas Systems
Septal Nuclei/metabolism
*Avoidance Learning/physiology
Behavior, Animal/physiology

@article {pmid41123407,
year = {2025},
author = {Xu, Z and Huang, Y and Dong, Y and An, X and Tong, Y and Li, M},
title = {Engineering broad-spectrum phage-resistant Escherichia coli via adaptive and programmable defense strategies.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0159625},
doi = {10.1128/aem.01596-25},
pmid = {41123407},
issn = {1098-5336},
support = {2022FY101100//Science & Technology Fundamental Resources Investigation Program/ ; 202423m10050004//Anhui Province Scientific and Technological Research Project/ ; 22322908D//S&T Program of Hebei/ ; 82341119//National Natural Science Foundation of China/ ; XK2025-05//Interdisciplinary Research Center of Beijing University of Chemical Technology/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/genetics/physiology/isolation & purification ; CRISPR-Cas Systems ; Genome, Viral ; Fermentation ; },
abstract = {UNLABELLED: Phage contamination, which impacts product quality and production efficiency, remains a major challenge in industrial fermentation. Although bacteria have evolved various defense systems to combat phage infection, these systems often suffer from narrow host specificity and limited efficacy. In this study, we isolated and characterized a novel lytic Escherichia coli phage, TR2, from a contaminated fermentation substrate. Its strong environmental stability, short latency period, and high lytic activity render it a significant threat to fermentation processes. Genomic sequencing revealed that phage TR2 has a linear, double-stranded DNA genome of 45,171 bp with a G+C content of 44% and 74 coding sequences. On the basis of the physiological characteristics and genomic features of this phage, we developed two strategies to generate phage-resistant E. coli strains: (i) selection of spontaneous mutations in bacterial surface receptors to prevent phage adsorption and infection and (ii) integration of an exogenous CRISPR/Cas9 system to confer sequence-specific immunity. Spontaneous mutation provides broad-spectrum resistance but at the cost of fitness and evolutionary stability, whereas CRISPR/Cas9 ensures long-term, programmable immunity with minimal growth defects. Importantly, both strategies successfully protected bacterial cultures from phage infection without compromising recombinant protein production, highlighting their potential for industrial application. Our findings provide a practical approach for mitigating phage contamination in industrial fermentation processes. This study also highlights the advantages and limitations of spontaneous mutations and natural phage defense systems, offering valuable insights for the design of more effective phage-resistant microbial platforms.

IMPORTANCE: Phage contamination is a significant challenge in industrial fermentation and severely impacts product quality and production efficiency. We systematically compared spontaneous mutation and CRISPR/Cas9-mediated immunity as two strategies for engineering phage-resistant E. coli strains. Both approaches effectively protected bacterial cultures from phage infection without compromising recombinant protein production, underscoring their potential for industrial applications. Notably, spontaneous mutation conferred broad-spectrum resistance but was associated with fitness costs and limited evolutionary stability. In contrast, CRISPR/Cas9-based immunity offered long-term, programmable protection with minimal growth impairment. By delineating the trade-offs between these two strategies, our work provides a framework for selecting tailored phage resistance solutions suited to diverse biomanufacturing scenarios.},
}

*Escherichia coli/virology/genetics
*Coliphages/genetics/physiology/isolation & purification
CRISPR-Cas Systems
Genome, Viral
Fermentation

@article {pmid40962686,
year = {2025},
author = {Chen, H and Fan, S and Chen, K and Wang, F and Lu, M and Wu, Y and Lu, H and Li, J},
title = {CRISPR-edited iPSCs reveal BSN gene mutations induce neuronal hyperexcitability via astrocyte lipid accumulation.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {22},
number = {6},
pages = {e00740},
doi = {10.1016/j.neurot.2025.e00740},
pmid = {40962686},
issn = {1878-7479},
mesh = {*Astrocytes/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Humans ; *Neurons/metabolism/physiology ; *Mutation/genetics ; *Lipid Metabolism/genetics/physiology ; Gene Editing/methods ; CRISPR-Cas Systems ; *Nerve Tissue Proteins/genetics ; Cells, Cultured ; Epilepsy/genetics ; },
abstract = {Mutations in the BSN gene, encoding the presynaptic protein Bassoon, are implicated in epilepsy, but their impact on astrocytes remains unclear. Using CRISPR/Cas9, we introduced patient-derived BSN mutations (p.M1903V and c.5672insCG) into human induced pluripotent stem cells (iPSCs) and differentiated them into astrocytes. We found that mutant astrocytes exhibited significant lipid accumulation, evidenced by elevated free cholesterol, reduced arginase activity, and increased lipid droplets. Proteomic analysis revealed upregulation of lipid metabolism regulators, such as APOE and FASN. Electrophysiological recordings showed impaired Kir4.1 potassium channel function, depolarized resting membrane potential, and increased capacitance in mutant astrocytes following kainic acid stimulation. Co-culture experiments with neurons demonstrated that BSN-mutant astrocytes led to reduced neurite outgrowth, elevated neuronal apoptosis, increased pro-inflammatory cytokines (IL-1β, TNF-α), and neuronal hyperexcitability. These findings demonstrate that BSN mutations disrupt astrocyte lipid homeostasis and impair neurosupportive functions, thereby driving neuronal hyperexcitability. This study establishes astrocytes as critical mediators of epilepsy pathogenesis in BSN-related disorders and highlights lipid metabolism as a potential therapeutic target.},
}

*Astrocytes/metabolism
*Induced Pluripotent Stem Cells/metabolism
Humans
*Neurons/metabolism/physiology
*Mutation/genetics
*Lipid Metabolism/genetics/physiology
Gene Editing/methods
CRISPR-Cas Systems
*Nerve Tissue Proteins/genetics
Cells, Cultured
Epilepsy/genetics

@article {pmid41242302,
year = {2025},
author = {Ishihara, K and Kitagawa, S and Adachi, N and Akutsu, M and Senda, T and Inanaga, H and Numata, T},
title = {Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152978},
doi = {10.1016/j.bbrc.2025.152978},
pmid = {41242302},
issn = {1090-2104},
abstract = {Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.},
}

@article {pmid41242144,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, JM and Jeong, H and Park, J and Kang, BH and Rho, D and Son, S and Kang, T and Jeong, KH},
title = {Nanoplasmonic real-time RT-RPA and CRISPR/Cas12a detection for rapid point-of-care molecular diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {294},
number = {},
pages = {118216},
doi = {10.1016/j.bios.2025.118216},
pmid = {41242144},
issn = {1873-4235},
abstract = {The rapid and precise detection of nucleic acids is crucial for effective disease diagnosis and management at the point-of-care (POC) level. Here we report a palm-sized plasmonic photothermal platform for real-time on-chip recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection. An ultrathin photothermal nanoplasmonic cavity (PNC) of Au nanoislands (AuNIs) and an aluminum reflector delivers uniform and efficient photothermal heating under white LED illumination. The configuration drives isothermal amplification and CRISPR-mediated cleavage in a single microchamber while a fluorescence microlens array (FMLA) camera records real-time emission. The compact platform detects the SARS-CoV-2 E gene in 25 min at 25.7 copies per cartridge and achieves 100 % concordance with RT-qPCR across 42 clinical samples. This all-in-one platform can offer a robust and cost-effective solution for molecular diagnostics, facilitating scalable and real-time testing of infectious diseases in decentralized POC settings.},
}

@article {pmid41241907,
year = {2026},
author = {Treichel, AJ and Bazzini, AA and Tornini, VA},
title = {Functional Testing of Microproteins in a Vertebrate Model of Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2992},
number = {},
pages = {183-201},
pmid = {41241907},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/metabolism ; Ribosomes/metabolism/genetics ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Micropeptides ; },
abstract = {While thousands of putative microproteins have been identified through ribosome profiling, reporter assays, and mass spectrometry-based methods, their functional testing has remained challenging. Advances in genome sequencing and CRISPR/Cas technologies enable the prioritization and testing of candidate microprotein functions for roles in development, for example, in the maternal-to-zygotic transition or in neurodevelopment. Here, we describe the functional testing of microproteins in vivo using a vertebrate model of early development, Danio rerio (zebrafish).},
}

Animals
*Zebrafish/genetics/embryology/metabolism
CRISPR-Cas Systems
*Zebrafish Proteins/genetics/metabolism
Ribosomes/metabolism/genetics
Gene Expression Regulation, Developmental
Embryonic Development/genetics
Micropeptides

@article {pmid41241835,
year = {2025},
author = {Zhang, J and Li, L and Zhu, Y and Qian, K and Xu, Q and Qin, Y and Wu, L},
title = {Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04524},
pmid = {41241835},
issn = {1520-6882},
abstract = {In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.},
}

@article {pmid41241617,
year = {2025},
author = {Kalogeropoulos, K and van Beljouw, SPB and Feldmann, D and van den Berg, DF and Brouns, SJJ},
title = {Proteases in bacteriophage defense systems and their potential in bioengineering.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2025.10.006},
pmid = {41241617},
issn = {0968-0004},
abstract = {Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.},
}

@article {pmid41220317,
year = {2025},
author = {Rayad, N and Chowdhury, EA and Meno-Tetang, GML},
title = {The Impact of QSP Modeling on the Design and Optimization of Gene Therapy Approaches.},
journal = {CPT: pharmacometrics & systems pharmacology},
volume = {14},
number = {11},
pages = {1760-1764},
doi = {10.1002/psp4.70131},
pmid = {41220317},
issn = {2163-8306},
mesh = {*Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Dependovirus/genetics ; Genetic Vectors ; Animals ; RNA, Messenger/administration & dosage/genetics ; Nanoparticles/administration & dosage ; CRISPR-Cas Systems ; *Models, Biological ; Lipids/chemistry ; Tissue Distribution ; Liposomes ; },
abstract = {Quantitative Systems Pharmacology (QSP) is increasingly utilized to support the design and translation of gene therapies. This perspective outlines the application of QSP modeling across three domains of gene therapy: mRNA-based therapeutics, adeno-associated virus (AAV) vectors, and genome editing systems. We highlight opportunities for dose optimization, biomarker interpretation, and mechanistic understanding, while addressing current limitations in model generalizability, data sparsity, and translational relevance. Examples include QSP platforms for lipid nanoparticle (LNP)-delivered mRNA, physiologically based pharmacokinetics (PBPK)-informed AAV biodistribution models, and CRISPR-Cas9-based editing systems. These case studies demonstrate QSP's value in de-risking development and personalizing therapies for rare and complex diseases.},
}

*Genetic Therapy/methods
Humans
Gene Editing/methods
Dependovirus/genetics
Genetic Vectors
Animals
RNA, Messenger/administration & dosage/genetics
Nanoparticles/administration & dosage
CRISPR-Cas Systems
*Models, Biological
Lipids/chemistry
Tissue Distribution
Liposomes

@article {pmid41191401,
year = {2025},
author = {Guo, Y and Xiong, Y and Tong, W and Huang, X and Xiong, Y},
title = {CRISPR/Cas12a-Based Dynamic Light Scattering Assay for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Listeria monocytogenes.},
journal = {Analytical chemistry},
volume = {97},
number = {45},
pages = {25232-25244},
doi = {10.1021/acs.analchem.5c04740},
pmid = {41191401},
issn = {1520-6882},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Dynamic Light Scattering/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Particle Size ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.},
}

*Listeria monocytogenes/isolation & purification/genetics
*CRISPR-Cas Systems
*Dynamic Light Scattering/methods
Limit of Detection
Gold/chemistry
Metal Nanoparticles/chemistry
Nucleic Acid Amplification Techniques
*Biosensing Techniques/methods
Particle Size
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41052028,
year = {2025},
author = {Khan, S and Chen, L and Chou, CL and Khundmiri, SJ and Knepper, MA},
title = {Inducible Avp knockout mouse line.},
journal = {American journal of physiology. Renal physiology},
volume = {329},
number = {6},
pages = {F784-F795},
doi = {10.1152/ajprenal.00340.2025},
pmid = {41052028},
issn = {1522-1466},
support = {ZIA-HL001285//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; ZIA-HL006129//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {Animals ; Mice, Knockout ; *Arginine Vasopressin/genetics/deficiency/metabolism ; Aquaporin 2/metabolism/genetics ; *Tamoxifen/pharmacology ; Mice ; Male ; *Kidney/metabolism/pathology ; Female ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Arginine vasopressin (AVP) is a peptide hormone synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies toward understanding AVP physiology relied heavily on Brattleboro rats, which have a spontaneous mutation in the Avp gene and lack circulating AVP. However, these rats are difficult to breed due to high neonatal death and behavioral issues, causing commercial breeders to stop production. To address this, we developed a mouse line with tamoxifen-inducible deletion of Avp. We used CRISPR/Cas9 to insert loxP sites into the Avp gene. These mice were then bred with mice expressing a tamoxifen-inducible Cre recombinase. The resulting conditional knockout mice (Avp[flx/flx]Cre[+]) are viable, fertile, and healthy before induction. Administration of tamoxifen in 8-12-wk-old mice successfully deleted Avp, as confirmed by Sanger sequencing. This deletion caused a significant decrease in urine osmolality, a hallmark of AVP deficiency. The kidney structure remained normal, with no signs of medullary atrophy. In addition, these mice exhibited a substantially decreased expression of the aquaporin 2 water channel (AQP2), which is involved in water reabsorption in the kidney inner medulla. We illustrate the use of this model by using RNA-seq to profile the consequences of Avp deletion on gene expression in the kidney. The curated RNA-seq data can be browsed, searched, or downloaded at https://esbl.nhlbi.nih.gov/Databases/AVP-KO/. In conclusion, we successfully created an inducible Avp knockout mouse line that has been made available to the research community. This model will be valuable for studying water balance regulation, polycystic kidney disease, and the neural, vascular, and metabolic functions of vasopressin.NEW & NOTEWORTHY We developed an inducible Avp knockout mouse line that will be shared with the research community and is likely to be useful for further study of the regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin.},
}

Animals
Mice, Knockout
*Arginine Vasopressin/genetics/deficiency/metabolism
Aquaporin 2/metabolism/genetics
*Tamoxifen/pharmacology
Mice
Male
*Kidney/metabolism/pathology
Female
Mice, Inbred C57BL
CRISPR-Cas Systems

@article {pmid41241097,
year = {2025},
author = {Hołubowicz, R and Gao, F and Du, SW and Menezes, CR and Zhang, J and Hołubowicz, MW and Chen, PZ and Armbrust, N and Geilenkeuser, J and Liu, DR and Jeffery Truong, DJ and Westmeyer, GG and Palczewska, G and Palczewski, K},
title = {Scalable purification enables high-quality virus-like particles for therapeutic translation.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110946},
doi = {10.1016/j.jbc.2025.110946},
pmid = {41241097},
issn = {1083-351X},
abstract = {Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.},
}

@article {pmid41240805,
year = {2025},
author = {Ghoreshi, ZA and Ali-Hassanzadeh, M and Mashayekhi-Sardoo, H and Askarpour, H and Arefinia, N},
title = {The identification of tuberculosis using CRISPR technique: A systematic review and meta-analysis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {2},
pages = {117183},
doi = {10.1016/j.diagmicrobio.2025.117183},
pmid = {41240805},
issn = {1879-0070},
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is essential for effective management and control of tuberculosis. The diagnostic challenge is particularly acute for extrapulmonary TB, which accounts for approximately 15-20% of cases and often presents with paucibacillary samples. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis across diverse sample types owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.

METHODS: A comprehensive search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to PRISMA guidelines. Quality was assessed using the Joanna Briggs comprehensive checklist. Data synthesis and analyses, including subgroup analyses, were performed with Meta-Disc 1.4, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.

RESULTS: From 341 identified studies, 13 met the inclusion criteria, encompassing 1,572 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.91 (95%CI: 0.89-0.92) and 0.97 (95%CI: 0.95-0.98), respectively. The pooled diagnostic odds ratio was 498.67 (95%CI: 255.1-974.7), with an AUC of 0.99 in the SROC curve, denoting excellent diagnostic accuracy. Subgroup analysis by sample type revealed that sputum samples achieved sensitivity and specificity of 0.92 (95%CI: 0.89-0.94) and 0.97 (95%CI: 0.94-0.99), while extrapulmonary samples (BALF, pus, CSF) showed comparable performance with sensitivity of 0.89 (95%CI: 0.84-0.93) and specificity of 0.98 (95%CI: 0.94-0.99).

CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB across both pulmonary and extrapulmonary samples, with notable variances across different CRISPR variants and methodological approaches.},
}

@article {pmid41240616,
year = {2025},
author = {Zong, W and Xie, S and Chu, H and Han, S and Zhang, X},
title = {Beyond traditional antibacterial agents: Novel approaches to combat resistant pathogens.},
journal = {European journal of medicinal chemistry},
volume = {302},
number = {Pt 2},
pages = {118362},
doi = {10.1016/j.ejmech.2025.118362},
pmid = {41240616},
issn = {1768-3254},
abstract = {As bacterial infections caused by antibiotic-resistant strains become increasingly prevalent, traditional antibacterial therapies face mounting challenges. These resilient pathogens not only complicate the treatment of common infections but also undermine the efficacy of therapies for major diseases. This growing threat underscores the urgent need for innovative therapeutic strategies. In recent years, the combinatorial use of antibacterial agents has emerged as a promising approach to enhance efficacy and combat resistant bacteria. This review first provides an overview of antibacterial classifications and their mechanisms of action against bacterial infections. It then explores two combined treatment strategies: antibacterial-antibacterial combinations and antibacterial-non-antibacterial pairings, alongside a drug delivery technology: antibacterial-loaded liposomes. Furthermore, we highlight emerging frontiers in antimicrobial strategies, including CRISPR-Cas technologies, AI-driven discovery platforms, nanomaterials beyond liposomes, microbiota-based therapies, and immunotherapeutic approaches. Finally, we offer a forward-looking perspective on the challenges and opportunities shaping the future of antibacterial development in the biomedical field.},
}

@article {pmid41178406,
year = {2025},
author = {Pei, C and Yan, B and Wang, Y and Chen, T and Du, K and Ma, L and Wang, J},
title = {TdT/Cas12a cascade amplification biosensor for sensitive ALP activity detection.},
journal = {The Analyst},
volume = {150},
number = {23},
pages = {5330-5337},
doi = {10.1039/d5an00938c},
pmid = {41178406},
issn = {1364-5528},
mesh = {Humans ; *Biosensing Techniques/methods ; *Alkaline Phosphatase/metabolism/analysis ; HeLa Cells ; CRISPR-Cas Systems ; Limit of Detection ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {This study presents a novel biosensor based on TdT and CRISPR-Cas12a, which integrates the catalytic activity of terminal deoxynucleotidyl transferase (TdT) with the trans-cleavage property of CRISPR-Cas12a to achieve ultra-sensitive biomolecular detection. The biosensor exhibited a broad linear detection range from 0 to 0.2 U L[-1] and a remarkably low detection limit of 1.7 × 10[-3] U L[-1], demonstrating high specificity and sensitivity. In practical validation, the biosensor successfully quantified alkaline phosphatase (ALP) activity in both cervical cancer cells and HeLa cell lysates, even at a dilution factor of up to 10[6]-fold. Its sensitivity allowed precise detection at the single-cell level. This technology offers a robust, simple, and cost-effective platform for cancer diagnosis, treatment monitoring, and enzyme inhibitor screening, while maintaining excellent detection performance in complex biological samples. This breakthrough establishes a foundation for serological tumor screening and early disease diagnosis, while also opening new avenues for enhanced cancer management and clinical translation, indicating significant potential in translational medicine.},
}

Humans
*Biosensing Techniques/methods
*Alkaline Phosphatase/metabolism/analysis
HeLa Cells
CRISPR-Cas Systems
Limit of Detection
*DNA Nucleotidylexotransferase/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41171167,
year = {2025},
author = {Tharmatt, A and Guha, S and Kumeria, T and Yadav, S and Mittal, A and Chitkara, D},
title = {Lipopolymeric Nanoplex-Mediated CRISPR/Cas9 Delivery for VEGF-A Knockdown in Psoriatic Angiogenesis.},
journal = {ACS applied bio materials},
volume = {8},
number = {11},
pages = {10137-10157},
doi = {10.1021/acsabm.5c01478},
pmid = {41171167},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; *Psoriasis/drug therapy/metabolism/genetics/pathology ; Humans ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists & inhibitors ; Animals ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; *Neovascularization, Pathologic/drug therapy/genetics ; Mice ; Materials Testing ; *Polymers/chemistry ; Gene Knockdown Techniques ; *Nanoparticles/chemistry ; Angiogenesis ; },
abstract = {Psoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.},
}

*CRISPR-Cas Systems
*Psoriasis/drug therapy/metabolism/genetics/pathology
Humans
*Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists & inhibitors
Animals
*Biocompatible Materials/chemistry/pharmacology/chemical synthesis
Particle Size
*Neovascularization, Pathologic/drug therapy/genetics
Mice
Materials Testing
*Polymers/chemistry
Gene Knockdown Techniques
*Nanoparticles/chemistry
Angiogenesis

@article {pmid40982615,
year = {2025},
author = {Kanwal, F and Aslam, A and Torriero, AAJ},
title = {Microalgae-based biodiesel: integrating AI, CRISPR and nanotechnology for sustainable biofuel development.},
journal = {Emerging topics in life sciences},
volume = {8},
number = {3},
pages = {131-143},
pmid = {40982615},
issn = {2397-8554},
mesh = {*Microalgae/metabolism/genetics ; *Biofuels ; *Nanotechnology/methods ; *Artificial Intelligence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
abstract = {Microalgae are a promising feedstock for biodiesel due to their rapid growth, high lipid content and ability to use non-arable land and wastewater. This review synthesises recent advances in artificial intelligence (AI)-driven strain optimisation, engineering, nanotechnology-assisted processing, and life cycle and technoeconomic insights to evaluate pathways for industrialisation. Over the past decade (2015-2024), genetic engineering and, more recently, AI-guided strain selection have improved lipid productivity by up to 40%. Cultivation advances, including hybrid photobioreactor-open pond systems and precision pH/CO2 control, have enhanced biomass yields while reducing costs. Innovation in lipid extraction, such as supercritical CO2 and microwave-assisted methods, now achieves >90% yields with lower toxicity, while magnetic nanoparticle-assisted harvesting and electroflocculation have reduced energy inputs by 20-30%. Life cycle analyses (net energy ratio ~2.5) and integration of high-value co-products (e.g. pigments and proteins) underscore the need to align biological innovations with techno-economic feasibility. This review uniquely integrates advances in AI, CRISPR and nanotechnology with life cycle and techno-economic perspectives, providing a comprehensive framework that links laboratory-scale innovation to industrial feasibility and positions microalgal biodiesel as a viable contributor to global decarbonisation strategies.},
}

*Microalgae/metabolism/genetics
*Biofuels
*Nanotechnology/methods
*Artificial Intelligence
*Clustered Regularly Interspaced Short Palindromic Repeats
*CRISPR-Cas Systems