@article {pmid40634673,
year = {2025},
author = {Asumadu, P and Guo, Z and Qi, S and Liu, C and Li, Y and Shi, Q and Kong, D and Ye, H and Fu, C and Wang, Z},
title = {Programmable DNA aptamer logic gates: from structural design to integrated systems for intelligent nanoscale biosensors.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {40634673},
issn = {1618-2650},
support = {BZ2022056//Caili Fu/ ; Science//Caili Fu/ ; Technology Project of Jiangsu Province//Caili Fu/ ; 32372417//Hua Ye/ ; National Natural Science Foundation of China//Hua Ye/ ; 1182921902//Dezhao Kong/ ; 32001804//Dezhao Kong/ ; Emerging Science//Dezhao Kong/ ; Technology Innovation Team Fu//Dezhao Kong/ ; National Natural Science Foundation of China//Dezhao Kong/ ; 22406069//Chang Liu/ ; National Natural Science Foundation of China//Chang Liu/ ; 1184882301//Qiaoqiao Shi/ ; Natural Science Foundation of the Jiangsu Higher E//Qiaoqiao Shi/ ; },
abstract = {DNA aptamer-based logic gates represent significant advances in molecular computing, enabling complex biological computations at the nanoscale. These systems leverage the unique programmable properties of DNA aptamers-short, single-stranded oligonucleotides with high specificity and binding affinity for diverse applications across fields such as clinical diagnostics, food/environmental monitoring, and targeted therapeutic delivery, garnering significant research interest in the past few decades. In this review, we first expand on the fundamentals of aptamers, including its SELEX process and post-SELEX modifications. We systematically examine the design principles and operation mechanisms of DNA aptamer-based logic gates, mainly AND, OR, INHIBIT and NOT as reported by researchers. Then, we highlight various logic gates based on different oligonucleotides spanning from intact and split aptamers to DNA origami architectures, DNA nanorobots, and G-quadruplex structural switches, bringing to light their applications across various fields. Recent innovations in multi-input/output gate cascades, CRISPR-Cas-integrated systems and signal amplification approaches are highlighted as key developments in DNA aptamer-based logic gates. Finally, we elucidate challenges relating to DNA aptamer-based systems such as aptamer performance, cross-reactivity in complex multi-input systems and the complexities of merging other systems to amplify output readability, among others, to the end that in addressing these challenges, we will be able to unlock the full potential of this system.},
}

@article {pmid40632881,
year = {2025},
author = {Thi Pham, N and Wang, CH and Chen, CH and Tseng, YT and Guthula, LS and Chau, LK and Yu, CY and Chen, YL},
title = {Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01035},
pmid = {40632881},
issn = {2379-3694},
abstract = {The highly prevalent single nucleotide polymorphism (SNP, rs671) of the aldehyde dehydrogenase (ALDH2) gene in Asian populations instigates various human pathologies and thus accentuates the urgent need for effective diagnostic tools. In this study, we present an ultrasensitive biosensing method by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with the fiber optic nanogold-linked sorbent assay (FONLISA) for precise SNP identification. This method leverages the sequence-specific recognition capability of the CRISPR/Cas system and the ultrahigh sensitivity via the dual signal enhancement mechanisms by integrating the trans-cleavage mechanism of Cas12a to amplify the signal from an activity reporter and the subsequent waveguide-enhanced nanoplasmonic absorption by a signaling reporter. In this method, Cas12a targets a double-stranded DNA from the ALDH2 SNP and then activates the degradation of the activity reporter, a free biotin-labeled single-stranded DNA probe (ssDNA[b]), by trans-cleavage. An unhybridized complementary single-stranded DNA probe (ssDNA[c]) labeled with a gold nanoparticle (AuNP) as the signaling reporter (AuNP@ssDNA[c]) is subsequently released and captured by the immobilized ssDNA[b] on the fiber core surface, resulting in a detectable nanoplasmonic absorption signal. The method also utilized an indispensable nanoplasmonic signal generator, carboxymethyl dextran-coated AuNP, to improve the preparation and bioconjugation processes. The CRISPR-FONLISA system demonstrates the ability to analyze the ALDH2 rs671 SNP from double-stranded DNA with a limit of detection of 71 aM. Furthermore, both cell lines and unamplified DNA extracted from blood samples were conducted to verify the system accuracy for ALDH2 rs671 SNP detection.},
}

@article {pmid40632864,
year = {2025},
author = {Yi, JY and Choi, H and Kim, M and Jeong, Y and Hahn, JS and Son, B and Park, HH and Sung, C},
title = {High-throughput multiplexed gene and cell doping analysis through CRISPR-Cas12a system integrated with blood direct PCR.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadv7234},
doi = {10.1126/sciadv.adv7234},
pmid = {40632864},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Doping in Sports ; *Polymerase Chain Reaction/methods ; *High-Throughput Screening Assays ; },
abstract = {Advancements in gene and cell therapies introduce "gene and cell doping," requiring efficient and sensitive detection methods. Here, we report a high-throughput multiplexed gene and cell doping analysis (HiMDA) using CRISPR-Cas12a system integrated with blood direct polymerase chain reaction (PCR). Blood direct PCR enables simultaneous amplification of multiple exogenous genes directly from whole-blood samples. Coupled with sequence-specific DNA recognition and fluorescence reporter system, HiMDA achieves multiplexed, on-target detection of doping genes and cells. Our results demonstrate HiMDA's feasibility with only 5 microliters of blood required for the entire 90-minute process. HiMDA exhibits exceptional sensitivity, detecting as few as 2.5 copies of doping target genes from blood-four times more sensitive than current anti-doping standards-and identifying in vivo doping up to 10 days. These findings highlight HiMDA's robust high-throughput, multiplexed capabilities, satisfying the sensitivity and selectivity demands of anti-doping research. HiMDA offers a flexible solution to meet future doping detection challenges.},
}

*CRISPR-Cas Systems/genetics
Humans
*Doping in Sports
*Polymerase Chain Reaction/methods
*High-Throughput Screening Assays

@article {pmid40632850,
year = {2025},
author = {Pal, P and Gao, S and Gao, H and Qin, X and Cella, M and Wang, Q and Shan, L},
title = {Establishment of a reverse genetics system for studying human immune functions in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadu1561},
doi = {10.1126/sciadv.adu1561},
pmid = {40632850},
issn = {2375-2548},
mesh = {Animals ; Humans ; Mice ; *Reverse Genetics/methods ; Antigens, CD34/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Fetal Blood/cytology ; Disease Models, Animal ; },
abstract = {Reverse genetics approaches in mice are widely used to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of disease pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood-derived CD34[+] cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34[+] cells recapitulate functional consequences of specific gene losses in the human immune system. Our approach enables targeted gene knockouts in humanized mice, offering a valuable tool for human gene function studies in vivo.},
}

Animals
Humans
Mice
*Reverse Genetics/methods
Antigens, CD34/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Fetal Blood/cytology
Disease Models, Animal

@article {pmid40632760,
year = {2025},
author = {Chao, A and Wang, J and Xiu, L and Bold, B and Ghonaim, AH and Chen, J and Hu, Q and Yin, K},
title = {CRISPR/Cas-Based Biosensing Strategies for Non-Nucleic Acid Contaminants in Food Safety: Status, Challenges, and Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c04162},
pmid = {40632760},
issn = {1520-5118},
abstract = {Non-nucleic acid targets (non-NATs), such as heavy metals, toxins, and pesticide residues, pose critical threats to food safety. Although CRISPR/Cas systems were initially developed for nucleic acid detection, recent advances have enabled their adaptation to non-NATs analysis by transducing target recognition into nucleic acid signals. Unlike previous reviews categorized by target type, this work establishes a mechanism-centric framework, systematically classifying non-NAT-to-nucleic acid signal conversion methodologies into three paradigms: (1) aptamer-based systems, (2) catalytic nucleic acid-based methods (e.g., DNAzymes), and (3) protein-mediated strategies (e.g., antibodies, transcription factors). When integrated with CRISPR/Cas, these systems achieve rapid, sensitive detection at picomolar (pM) levels without relying on chromatographic or spectroscopic instruments. Furthermore, we critically discuss challenges, including the limited diversity of recognition elements, inefficient signal conversion, and inflexible signal outputs, proposing solutions including synthetic-biology-driven bioreceptor design and artificial-intelligence-based data analysis. By bridging mechanistic principles with applications in complex food matrices, this review provides actionable insights to advance CRISPR-based tools for rapid, on-site, food safety monitoring.},
}

@article {pmid40632661,
year = {2025},
author = {Wang, S and Zheng, J and Zhang, X and Zhao, M and Li, J and Su, M and Qiu, W},
title = {Genome-Wide CRISPR-Cas9 Knockout Screening Identifies Genes Modulating Cisplatin-Induced Cytotoxicity in Renal Proximal Tubule Epithelial Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {13},
pages = {e70780},
doi = {10.1096/fj.202402401RR},
pmid = {40632661},
issn = {1530-6860},
support = {5202009//Natural Science Foundation of Beijing Municipality/ ; 5192022//Natural Science Foundation of Beijing Municipality/ ; 81870196//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Cisplatin/toxicity/pharmacology/adverse effects ; *Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology ; *CRISPR-Cas Systems/genetics ; *Epithelial Cells/drug effects/metabolism/pathology ; Humans ; *Antineoplastic Agents/toxicity ; Animals ; Gene Knockout Techniques ; },
abstract = {Cisplatin is widely used as a first-line chemotherapy drug for various cancers. However, cisplatin-induced nephrotoxicity (CIN) greatly restricts its application. Renal proximal tubular epithelial cells (RPTECs) can be extensively damaged during CIN. However, it still lacks an ideal method to prevent CIN, because the mechanism and therapeutic targets of CIN remain largely unclear. In the present study, we used a genome-scale CRISPR-Cas9 knock-out method to functionally screen key genes of cisplatin-induced RPTEC injury. We found 815 genes significantly enriched (p < 0.05) from positive selection screening strategy, which may synergistically enhance cisplatin cytotoxicity in RPTECs. Importantly, we identified ERAP2 as a novel molecule associated with CIN. We found that the expression of ERAP2 in RPTECs was significantly up-regulated by cisplatin. Data from CCK-8 assay and flow cytometry showed that inhibition of ERAP2 alleviated cisplatin-induced RPTEC injury. Furthermore, RNA-seq and qPCR results revealed that three necroptosis-associated genes, PLA2G4C, HIST1H2AC, and HIST1H2AM, were downregulated following ERAP2 inhibition, suggesting that ERAP2 may be a novel therapeutic target of CIN through the modulation of necroptosis pathway.},
}

*Cisplatin/toxicity/pharmacology/adverse effects
*Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology
*CRISPR-Cas Systems/genetics
*Epithelial Cells/drug effects/metabolism/pathology
Humans
*Antineoplastic Agents/toxicity
Animals
Gene Knockout Techniques

@article {pmid40632351,
year = {2025},
author = {Lokya, V and Singh, S and Chaudhary, R and Jangra, A and Tiwari, S},
title = {Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {84},
pmid = {40632351},
issn = {1573-5028},
support = {BIRAC/TechTransfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; Core research grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; },
mesh = {*Gene Editing/methods/trends ; *Crops, Agricultural/genetics/growth & development ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Genome editing tools have revolutionized plant biology research offering unparalleled applications for genome manipulation and trait improvement in crops. Adopting such advanced biotechnological tools is inevitable to meet increasing global food demand and address challenges in food production, including (a)biotic stresses and inadequate nutritional value. Despite reliance on conventional genetic manipulation methods, the CRISPR-Cas-mediated genome editing toolbox allows precise modification of DNA/RNA in a target organism's genome. So far, CRISPR-Cas has been widely used to enhance yield, quality, stress tolerance, and nutritional value in various food crops. However, challenges such as reagent delivery in suitable explants, precise editing with minimal off-target effect, and generating transgene-free plants persist as major bottlenecks in most plant species. Components of CRISPR-Cas construct mainly Cas, guide RNA (gRNA), and selectable marker genes are often integrated into the host genome, which raises regulatory concerns. However, adapting advanced gene-editing strategies, including high-efficiency Cas endonucleases, DNA-independent RNP delivery, morphogenetic regulators, and grafting-mediated editing, are paving the way for transgene-free crop improvement while easing biosafety regulations. Further, regulatory frameworks for genome-edited crops vary globally, with several countries accepting them and others debating their legal status. Hence, the disparity in global regulatory guidelines of genome editing curbs commercialization. The current review highlights the emerging CRISPR-mediated tools or methods and their applications in developing transgene-free designer crops to harness the benefits of advanced genome manipulation.},
}

*Gene Editing/methods/trends
*Crops, Agricultural/genetics/growth & development
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
*Genome, Plant
Transgenes

@article {pmid40632173,
year = {2025},
author = {Thomas, L and Abraham, T},
title = {Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step towards antivirulence therapy.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {8},
pages = {110},
pmid = {40632173},
issn = {1572-9699},
support = {363661//University Grants Commission/ ; },
mesh = {*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; *Iron/metabolism ; *Fimbriae, Bacterial/metabolism/genetics ; Escherichia coli Proteins/genetics/metabolism ; Escherichia coli Infections/microbiology ; Virulence Factors/genetics/metabolism ; Virulence/genetics ; Molecular Docking Simulation ; Humans ; Fimbriae Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes-iucD, involved in aerobactin-mediated iron uptake, and papC, encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated iucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.},
}

*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity
*CRISPR-Cas Systems
*Iron/metabolism
*Fimbriae, Bacterial/metabolism/genetics
Escherichia coli Proteins/genetics/metabolism
Escherichia coli Infections/microbiology
Virulence Factors/genetics/metabolism
Virulence/genetics
Molecular Docking Simulation
Humans
Fimbriae Proteins/genetics/metabolism
Gene Editing

@article {pmid40631558,
year = {2025},
author = {He, C and Zhu, W and Zhang, X and Wu, W and Tan, J and Li, B and Huang, D and Chen, Y and Xiang, Z and Huang, L and Gong, L},
title = {Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.},
journal = {Journal of medical virology},
volume = {97},
number = {7},
pages = {e70460},
doi = {10.1002/jmv.70460},
pmid = {40631558},
issn = {1096-9071},
support = {//This study is financially supported by The President Foundation of Nanfang Hospital, Southern Medical University (grant number 2023B036); National Science and Technology Infrastructure of China (Project No. National Pathogen Resource Center-NPRC-32); The CAMS Innovation Fund for Medical Sciences (grant number 2021-I2M-1-039); National Key R&D Program of China, grant number (grant number 2022YFC2303400); The Guangdong Basic and Applied Basic Research Foundation (grant number 2023A1515140080); Dongguan Social development technology projects Science and Technology Planning Project (grant number 20231800940792)./ ; },
mesh = {*CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; *Molecular Diagnostic Techniques/methods ; *Orthohantavirus/isolation & purification/genetics ; RNA, Viral/genetics ; *Hantavirus Infections/diagnosis/virology ; *Hantaan virus/genetics/isolation & purification ; DNA Primers/genetics ; },
abstract = {In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.},
}

*CRISPR-Cas Systems
Sensitivity and Specificity
Humans
*Molecular Diagnostic Techniques/methods
*Orthohantavirus/isolation & purification/genetics
RNA, Viral/genetics
*Hantavirus Infections/diagnosis/virology
*Hantaan virus/genetics/isolation & purification
DNA Primers/genetics

@article {pmid40630641,
year = {2025},
author = {Pan, K and Zeng, A and Ruan, X and Mo, X and Shen, B and Zhao, J and Zhou, Y},
title = {The apicoplast localized isocitrate dehydrogenase is needed for de novo fatty acid synthesis in the apicoplast of Toxoplasma gondii.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1542122},
pmid = {40630641},
issn = {2235-2988},
mesh = {*Toxoplasma/enzymology/growth & development/genetics/metabolism ; *Isocitrate Dehydrogenase/metabolism/genetics ; Animals ; *Fatty Acids/biosynthesis ; *Apicoplasts/enzymology/metabolism/genetics ; Mice ; Virulence ; CRISPR-Cas Systems ; Gene Deletion ; NADP/metabolism ; Glucose/metabolism ; },
abstract = {Toxoplasma gondii (T. gondii), an apicomplexan parasite, infects a wide range of warm-blooded animals and poses significant risks to human health. The fatty acid synthesis II (FASII) pathway in the apicoplast, which is the major source of fatty acids in parasites, is considered a potential drug target. The apicoplast also harbors some enzymes of central carbon metabolism, which are crucial for its survival, but their biological roles remain unclear. In this study, we focused on apicoplast-localized isocitrate dehydrogenase 1 (ICDH1) and deleted it using CRISPR-Cas9 technology. The Δicdh1 mutant tachyzoites displayed markedly impaired growth kinetics, with further suppression under serum-deprived conditions. However, this deletion did not affect the viability or virulence of the Δicdh1 mutant in mice. NADPH, a product of ICDH1-mediated decarboxylation of isocitrate, is an essential cofactor for fatty acid synthesis. Using [13]C6 glucose as a metabolic carbon source, we showed that the mutant strains had reduced incorporation of glucose-derived carbons into medium-chain length fatty acids (C14:0 and C16:0). Additionally, the growth of the mutant was partially restored by supplementation with exogenous C14:0 and C16:0 fatty acids. These results indicate that ICDH1 deletion affects the FASII pathway and parasite growth. Consistent with previous studies, this study confirms that T. gondii has metabolic flexibility in the apicoplast that allows it to acquire fatty acids through various pathways.},
}

*Toxoplasma/enzymology/growth & development/genetics/metabolism
*Isocitrate Dehydrogenase/metabolism/genetics
Animals
*Fatty Acids/biosynthesis
*Apicoplasts/enzymology/metabolism/genetics
Mice
Virulence
CRISPR-Cas Systems
Gene Deletion
NADP/metabolism
Glucose/metabolism

@article {pmid40629383,
year = {2025},
author = {Gou, F and Liu, D and Gong, C and Wang, K and Wang, X and Chen, Y and Liu, Q and Tian, C},
title = {Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {160},
pmid = {40629383},
issn = {1475-2859},
support = {2023YFC3402300//National Key Research & Developmental Program of China/ ; U22A20441//National Natural Science Foundation of China/ ; XDA0510300//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Aspergillus niger/genetics/metabolism/enzymology ; *Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism ; Recombinant Proteins/genetics/biosynthesis/metabolism ; CRISPR-Cas Systems ; Industrial Microbiology ; Genetic Engineering ; },
abstract = {BACKGROUND: Aspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.

RESULTS: In this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48-72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 - 1328 U/mL, ~ 1627. 43 - 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.

CONCLUSIONS: Our results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.},
}

*Aspergillus niger/genetics/metabolism/enzymology
*Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism
Recombinant Proteins/genetics/biosynthesis/metabolism
CRISPR-Cas Systems
Industrial Microbiology
Genetic Engineering

@article {pmid40586242,
year = {2025},
author = {Sreekanth, V and Jan, M and Zhao, KT and Lim, D and Siriwongsup, S and Davis, JR and McConkey, M and Kovalcik, V and Barkal, S and Law, BK and Fife, J and Tian, R and Vinyard, ME and Becerra, B and Kampmann, M and Sherwood, RI and Pinello, L and Liu, DR and Ebert, BL and Choudhary, A},
title = {A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {27},
pages = {23844-23856},
doi = {10.1021/jacs.5c06230},
pmid = {40586242},
issn = {1520-5126},
mesh = {Half-Life ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism ; Ubiquitination ; },
abstract = {Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.},
}

Half-Life
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism
Ubiquitination

@article {pmid40577080,
year = {2025},
author = {Butti, P and Bellusci, F and Brambilla, E and Branduardi, P},
title = {Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf032},
pmid = {40577080},
issn = {1567-1364},
support = {//University of Milano-Bicocca/ ; CN_00000033//Italian Ministry of University and Research/ ; H43C22000530001//Italian Ministry of University and Research/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Gene Editing/methods ; *Genome, Fungal ; },
abstract = {A large variety of synthetic biology toolkits for the introduction of multiple expression cassettes is available for Saccharomyces cerevisiae. Unfortunately, none of these tools is designed to allow the modification - exchange or removal - of the cassettes already integrated into the genome in a standardized way. The application of the modularity principle therefore ends to the steps preceding the final host engineering, making microbial cell factories construction stiff and strictly sequential. In this work, we describe a system that easily allows CRISPR-mediated swapping or removal of previously integrated cassettes, thus bringing the modularity to the strain level, enhancing the possibility of modifying existing strains with a reduced number of steps. In the system, each cassette is tagged with specific barcodes, which can be used as targets for CRISPR nucleases (Cas9 and Cas12a), allowing the excision of the construct from the genome and its substitution with another expression cassette or the restoration of the wild type locus in one single standardized step. The system has been applied to the previously developed Easy-MISE toolkit and tested by swapping fluorescent protein expression cassettes with an efficiency of ∼90% quantified by PCR and flow cytometry.},
}

*Saccharomyces cerevisiae/genetics
CRISPR-Cas Systems
*Synthetic Biology/methods
*Gene Editing/methods
*Genome, Fungal

@article {pmid40552742,
year = {2025},
author = {Petroulia, S and Hockemeyer, K and Tiwari, S and Berico, P and Shamloo, S and Banijamali, SE and Vega-Saenz de Miera, E and Gong, Y and Thandapani, P and Wang, E and Schloßhauer, JL and Tsirigos, A and Osman, I and Aifantis, I and Imig, J},
title = {Uncovering Novel lncRNAs Linked to Melanoma Growth and Migration with CRISPR Inhibition Screening.},
journal = {Cancer research communications},
volume = {5},
number = {7},
pages = {1102-1118},
doi = {10.1158/2767-9764.CRC-24-0416},
pmid = {40552742},
issn = {2767-9764},
mesh = {Humans ; *Melanoma/genetics/pathology ; *RNA, Long Noncoding/genetics ; Cell Movement/genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; *Skin Neoplasms/genetics/pathology ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Apoptosis/genetics ; },
abstract = {UNLABELLED: Melanoma being one of the most common and deadliest skin cancers has been increasing since the past decade. Patients at advanced stages of the disease have very poor prognoses, as opposed to at the earlier stages. Nowadays, the standard of care of advanced melanoma is resection, followed by immune checkpoint inhibition-based immunotherapy. However, a substantial proportion of patients either do not respond or develop resistance. This underscores a need for novel approaches and therapeutic targets as well as a better understanding of the mechanisms of melanoma pathogenesis. Long noncoding RNAs (lncRNA) comprise a poorly characterized class of functional players and promising targets in promoting malignancy. Certain lncRNAs have been identified to play integral roles in melanoma progression and drug resistance; however, systematic screens to uncover novel functional lncRNAs are scarce. In this study, we profile differentially expressed lncRNAs in patient-derived short-term metastatic cultures and BRAF-MEK inhibition-resistant cells. We conduct a focused growth-related CRISPR inhibition screen of overexpressed lncRNAs, validate, and functionally characterize lncRNA hits with respect to cellular growth, invasive capacities, and apoptosis in vitro as well as the transcriptomic impact of our lead candidate the novel lncRNA XLOC_030781. In sum, we extend the current knowledge of ncRNAs and their potential relevance in melanoma.

SIGNIFICANCE: LncRNAs have emerged as novel players in regulating many cellular aspects also in melanoma. The number of functional significances of most lncRNAs remains elusive. We provide a comprehensive strategy to identify functionally relevant lncRNAs in melanoma by combining expression profiling with CRISPR inhibition growths screens. Our results broaden the characterized lncRNAs as potential targets for future therapeutic applications.},
}

Humans
*Melanoma/genetics/pathology
*RNA, Long Noncoding/genetics
Cell Movement/genetics
Cell Line, Tumor
Cell Proliferation/genetics
*Skin Neoplasms/genetics/pathology
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Apoptosis/genetics

@article {pmid40482807,
year = {2025},
author = {Geiger, AB and Kennedy, JG and Staker, LG and Wensel, TG and Casson, RJ and Thomas, PQ},
title = {Shining light on CRISPR/Cas9 therapeutics for inherited retinal diseases.},
journal = {Progress in retinal and eye research},
volume = {107},
number = {},
pages = {101376},
doi = {10.1016/j.preteyeres.2025.101376},
pmid = {40482807},
issn = {1873-1635},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Retinal Diseases/therapy/genetics ; Animals ; },
abstract = {Inherited retinal diseases (IRDs), such as retinitis pigmentosa, are a heterogenous group of genetic eye diseases characterized by degeneration of photoreceptors. They are the leading cause of blindness in the working age population in high-income countries and are an ideal target for the expanding gene editing tool kit, including rapidly evolving CRISPR/Cas9 technology. In this review, we provide a comprehensive analysis of CRISPR/Cas9 technologies currently being explored as therapeutic interventions for IRDs. Given the challenges posed by the growing complexity and size of gene editing systems, the delivery of these therapeutics to the retina has necessitated innovative approaches. We review current delivery methods, including nanoparticles, virus-like particles and traditional viral vectors, highlighting their advantages and limitations. This review underscores the potential transformative impact of gene editing on genetic disease management, emphasising that advancements in these technologies, coupled with improved pre-clinical models, bring clinically safe and effective treatments for IRDs within view.},
}

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

@article {pmid40434115,
year = {2025},
author = {Wiechert, J and Badia Roigé, B and Dohmen-Olma, D and Hindra, and Zhang, X and Stella, RG and Elliot, MA and Frunzke, J},
title = {CRISPR/dCas-mediated counter-silencing: reprogramming dCas proteins into antagonists of xenogeneic silencers.},
journal = {mBio},
volume = {16},
number = {7},
pages = {e0038225},
doi = {10.1128/mbio.00382-25},
pmid = {40434115},
issn = {2150-7511},
support = {458090666//Deutsche Forschungsgemeinschaft/ ; 162340/CAPMC/CIHR/Canada ; //Jürgen Manchot Stiftung/ ; },
mesh = {*CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; *Streptomyces/genetics ; *Gene Silencing ; *Gene Expression Regulation, Bacterial ; *Corynebacterium glutamicum/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {UNLABELLED: Lsr2-like nucleoid-associated proteins function as xenogeneic silencers (XSs) inhibiting expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. Interference by transcription factors can lead to counter-silencing of XS target promoters, but relief of this repression typically requires promoter engineering. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach by using nuclease-deficient dCas enzymes to counteract the Lsr2-like XS protein CgpS in Corynebacterium glutamicum or Lsr2 in Streptomyces venezuelae. Systematic in vivo reporter studies with dCas9 and dCas12a and various guide RNAs revealed effective counter-silencing of different CgpS target promoters in response to guide RNA/dCas DNA binding, independent of promoter sequence modifications. The most prominent CRISPRcosi effect was observed when targeting the CgpS nucleation site, an effect that was also seen in S. venezuelae when targeting a known Lsr2 nucleation site within the chloramphenicol biosynthesis gene cluster. Analyzing the system in C. glutamicum strains lacking the XS protein CgpS revealed varying strengths of counteracting CRISPR interference effects based on the target position and strand. Genome-wide transcriptome profiling in single-guide RNA/dCas9 co-expressing C. glutamicum wild-type strains revealed high counter-silencing specificity with minimal off-target effects. Thus, CRISPRcosi provides a promising strategy for the precise upregulation of XS target genes with significant potential for studying gene networks as well as for developing applications in biotechnology and synthetic biology.

IMPORTANCE: Lsr2-like nucleoid-associated proteins act as xenogeneic silencers (XSs), repressing the expression of horizontally acquired, adenine-thymine-rich DNA in actinobacteria. The targets of Lsr2-like proteins are very diverse, including prophage elements, virulence gene clusters, and biosynthetic gene clusters. Consequently, the targeted activation of XS target genes is of interest for fundamental research and biotechnological applications. Traditional methods for counter-silencing typically require promoter modifications. In this study, we developed a novel clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-mediated counter-silencing (CRISPRcosi) approach, utilizing nuclease-deficient dCas enzymes to counteract repression by Lsr2-like proteins in Corynebacterium glutamicum and Streptomyces venezuelae. The strongest effect was observed when targeting the Lsr2 nucleation site. Genome-wide transcriptome profiling revealed high specificity with minimal off-target effects. Overall, CRISPRcosi emerges as a powerful tool for the precise induction of genes silenced by xenogeneic silencers, offering new opportunities for exploring gene networks and advancing biotechnological applications.},
}

*CRISPR-Cas Systems
Promoter Regions, Genetic
*Bacterial Proteins/genetics/metabolism
*Streptomyces/genetics
*Gene Silencing
*Gene Expression Regulation, Bacterial
*Corynebacterium glutamicum/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39915620,
year = {2025},
author = {Pagliaro, A and Andreatta, F and Finger, R and Artegiani, B and Hendriks, D},
title = {Generation of human fetal brain organoids and their CRISPR engineering for brain tumor modeling.},
journal = {Nature protocols},
volume = {20},
number = {7},
pages = {1846-1883},
pmid = {39915620},
issn = {1750-2799},
mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain Neoplasms/genetics/pathology ; *Brain/embryology/cytology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Fetus/cytology ; },
abstract = {The developing human brain displays unique features that are difficult to study in animal models. Current in vitro models based on human brain tissue face several challenges, including the limited cellular heterogeneity in two- or three-dimensional neural stem cell cultures, while tissue slice cultures suffer from short survival. We recently established culture conditions to derive organoid cultures directly from human fetal brain tissue by preserving tissue integrity, which can be long-term expanded and display cellular heterogeneity and complex organization. In this Protocol, we describe detailed procedures to establish human fetal brain organoids (FeBOs) that broadly retain regional characteristics, along with procedures for their passaging and characterization. In addition, we describe genome engineering approaches applied to FeBOs to generate mutant FeBO lines that serve as versatile bottom-up brain cancer models. Lastly, we exemplify various downstream applications applicable to both healthy and mutant FeBOs. Scientists with experience in tissue culture can expect the establishment of human FeBO cultures to take 2-3 weeks, while genome engineering of FeBOs takes 2-4 months.},
}

Humans
*Organoids/cytology/metabolism
*Brain Neoplasms/genetics/pathology
*Brain/embryology/cytology
*CRISPR-Cas Systems
*Gene Editing/methods
Fetus/cytology

@article {pmid40627177,
year = {2025},
author = {Azani, A and Sharafi, M and Doachi, R and Akbarzadeh, S and Lorestani, P and Haji Kamanaj Olia, A and Zahed, Z and Gharedaghi, H and Ghasrsaz, H and Foroozand, H and Rahimi, H and Tahmasebi, S and Behfar, Q},
title = {Applications of CRISPR-Cas9 in mitigating cellular senescence and age-related disease progression.},
journal = {Clinical and experimental medicine},
volume = {25},
number = {1},
pages = {237},
pmid = {40627177},
issn = {1591-9528},
mesh = {Humans ; *Cellular Senescence/genetics ; *CRISPR-Cas Systems ; *Aging/genetics ; Epigenesis, Genetic ; *Gene Editing/methods ; Disease Progression ; Animals ; Telomerase/genetics/metabolism ; },
abstract = {Aging is a multifaceted process influenced by many elements. During cell division, the repetitive DNA sequences at the ends of chromosomes called telomeres protect them from degradation. Telomeres shorten alongside each cell division, eventually contributing to cellular senescence and aging. Telomerase as an enzyme has a role in the maintenance of telomere length. Reduced function of telomerase is linked to acceleration of aging and age-related diseases. By affecting cellular function, mutations in particular genes can cause aging. Genes involved in DNA repair, cellular metabolism, and inflammation play the key roles in this process. Accumulated mutations result in cellular dysfunction and age-related diseases over time. Epigenetic changes are the modifications that impact gene expression without altering the DNA sequence. Lifestyle factors (diet, exercise, stress) and environmental influences (toxins, trauma) can cause epigenetic alterations. DNA methylation as well as histone modifications are examples of epigenetic alterations. They influence how cells work and are essential to the aging process. Understanding these molecular mechanisms is essential for developing interventions to promote healthy aging and prevent age-related diseases. This paper explores the potential of CRISPR/Cas9 as a gene-editing tool to target these mechanisms and mitigate age-related conditions, ultimately enhancing longevity and quality of life.},
}

Humans
*Cellular Senescence/genetics
*CRISPR-Cas Systems
*Aging/genetics
Epigenesis, Genetic
*Gene Editing/methods
Disease Progression
Animals
Telomerase/genetics/metabolism

@article {pmid40627029,
year = {2025},
author = {M, KR and C, SD},
title = {Recent insights into actinobacteria research in antimicrobial resistance: a review.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {683},
pmid = {40627029},
issn = {1573-4978},
mesh = {*Actinobacteria/genetics/metabolism/drug effects ; *Anti-Bacterial Agents/pharmacology ; Multigene Family ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Streptomyces/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Biosynthetic Pathways/genetics ; },
abstract = {Antimicrobial resistance (AMR) has emerged as a global health crisis, taking 4.71 million lives in the year 2021 and posing significant challenges to healthcare systems. Actinobacteria, particularly Streptomyces sp., are a well-established source of bioactive secondary metabolites, including antibiotics such as polyketides, aminoglycosides, and macrolides with activity against multidrug-resistant (MDR) bacteria. However, only 10% of the antibiotic genes are expressed, and others are silent in cryptic biosynthetic gene clusters (BGCs) that remain inactive under standard laboratory conditions. Advances in genome mining, bioinformatics tools like antiSMASH, and molecular techniques such as CRISPR-Cas have facilitated the identification of these clusters. Furthermore, innovative strategies such as co-culturing and HDAC inhibitors have shown promise in activating cryptic biosynthetic pathways to combat emerging antimicrobial resistance. Despite these advancements, the rapid evolution of resistance requires continuous research and global collaboration to ensure a sustainable pipeline of effective antibiotics. This review provides insight into actinobacteria-derived antibiotics, resistance mechanisms, and emerging biotechnological interventions to address the AMR crisis, underscoring the urgent need for multidisciplinary antibiotic discovery and stewardship efforts.},
}

*Actinobacteria/genetics/metabolism/drug effects
*Anti-Bacterial Agents/pharmacology
Multigene Family
Humans
*Drug Resistance, Multiple, Bacterial/genetics
Streptomyces/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Biosynthetic Pathways/genetics

@article {pmid40626824,
year = {2025},
author = {Özdemir, BH},
title = {Navigating Immunological Barriers in Xenotransplantation: Recent Advances and Promising Strides.},
journal = {Experimental and clinical transplantation : official journal of the Middle East Society for Organ Transplantation},
volume = {23},
number = {6},
pages = {421-430},
doi = {10.6002/ect.2023.0351},
pmid = {40626824},
issn = {2146-8427},
mesh = {Humans ; Animals ; *Transplantation, Heterologous/adverse effects ; *Graft Rejection/immunology/prevention & control/genetics ; Animals, Genetically Modified ; Swine ; *Immunity, Humoral ; *Graft Survival ; *Antibodies, Heterophile/immunology/genetics/blood ; *Antigens, Heterophile/immunology/genetics ; Immunity, Cellular ; Treatment Outcome ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The review introduces the challenges and potential solutions in xenotransplantation, focusing on pig-to-human organ transplant. Xenotransplantation, mainly with the use of pig organs, is a promising solution because of the reproductive capacity, size, and physiological resemblance of pigs to humans. However, immunological barriers, especially humoral and cellular immune responses, pose substantial challenges. The humoral immune response, involving antibodies targeting xenoantigens, is a substantial barrier. Anti-α-galactose antibodies, targeting α-Gal epitopes, are crucial in hyperacute rejection and acute humoral xenograft rejection. Genetic modifications, including CRISPR/Cas9 technology, aim to eliminate xenoantigens like α-Gal, potentially overcoming these challenges. This review discusses the use of genetically modified pigs for xenotransplantation, emphasizing the removal of xenoantigens, expression of human complement regulatory proteins, and transgenic expres-sion of human regulatory factors. Recent advancements, such as the world's first porcine-to-human heart transplant, highlight the potential of genetic manipu-lation in overcoming immune rejection barriers.},
}

Humans
Animals
*Transplantation, Heterologous/adverse effects
*Graft Rejection/immunology/prevention & control/genetics
Animals, Genetically Modified
Swine
*Immunity, Humoral
*Graft Survival
*Antibodies, Heterophile/immunology/genetics/blood
*Antigens, Heterophile/immunology/genetics
Immunity, Cellular
Treatment Outcome
CRISPR-Cas Systems
Gene Editing

@article {pmid40626243,
year = {2025},
author = {Mitousis, L and Musiol-Kroll, E and Wohlleben, W},
title = {CRISPR-Cas in actinomycetes: still a lot to be discovered.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf010},
pmid = {40626243},
issn = {2633-6693},
abstract = {Actinomycetes are important producers of valuable natural products that are applied in medicine or industry. The enzymes necessary for the synthesis of those compounds are encoded in biosynthetic gene clusters (BGCs) in the genome. However, the discovery of new natural products or the improvement of production levels can be hindered by difficulties in genetic manipulation, since standard methods often do not or not efficiently work in actinomycetes. One possible explanation for this could be the presence of nucleic acid defense systems such as CRISPR-Cas. Even though there is a lot of research published about CRISPR-Cas systems in general, the knowledge about the function of CRISPR-Cas in actinomycetes is very limited. Based on sequence data it is known that CRISPR-Cas systems occur in around half of all sequenced actinobacterial genomes. Moreover, in silico analyses of those systems have led to the discovery of new subtypes. The few examples of experimental evidence of CRISPR-Cas activity in vivo or in vitro, however, point to some special features, regarding crRNA maturation or life-cycle dependent CRISPR-Cas activity. This short review draws attention to this neglected research area and highlights the available data about CRISPR-Cas in actinomycetes.},
}

@article {pmid40364716,
year = {2025},
author = {Cahn, JKB and Ludwicki, H and Shingler, J and Gulvin, S and Zhang, Y and Kristopeit, A and Ton, C and Winters, MA and Wagner, JM and Moore, J},
title = {CRISPR-Editing of the Vero Cell Line Improves Processability of Live Virus Vaccines by Enabling Targeted Proteolysis of Fibronectin.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {8},
pages = {2082-2092},
doi = {10.1002/bit.29028},
pmid = {40364716},
issn = {1097-0290},
mesh = {Animals ; Chlorocebus aethiops ; Vero Cells ; *Gene Editing/methods ; *Fibronectins/genetics/metabolism ; *CRISPR-Cas Systems ; *Viral Vaccines/genetics/metabolism ; Proteolysis ; Vaccines, Attenuated/genetics ; },
abstract = {Removal of host cell components is a significant cost driver in the production of live virus vaccines. Filtration processes such as tangential flow filtration can be effective in this capacity by leveraging the relative size difference between viral particles and host proteins; however, filtration membranes can be fouled by larger proteins, particularly those of the extracellular matrix. In this study, we used CRISPR editing to insert the recognition element of the highly-selective TEV protease into various positions of the gene encoding fibronectin in the genome of the Vero cell line, a common platform for viral production. By screening edited cell lines, we identified a promising candidate line in which fibronectin could be effectively removed by treating with the protease during processing, eliminating filter fouling and allowing for viral purification without the need for costly chromatography steps.},
}

Animals
Chlorocebus aethiops
Vero Cells
*Gene Editing/methods
*Fibronectins/genetics/metabolism
*CRISPR-Cas Systems
*Viral Vaccines/genetics/metabolism
Proteolysis
Vaccines, Attenuated/genetics

@article {pmid40345583,
year = {2025},
author = {Flores-Arenas, C and Malekos, E and Montano, C and Covarrubias, S and Sudek, L and Dempsey, V and Huynh, V and Katzman, S and Carpenter, S},
title = {CRISPRi screen uncovers lncRNA regulators of human monocyte growth.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {6},
pages = {110204},
pmid = {40345583},
issn = {1083-351X},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Monocytes/cytology/metabolism ; *Cell Proliferation ; *CRISPR-Cas Systems ; THP-1 Cells ; Interferon Regulatory Factors/genetics/metabolism ; Apoptosis ; },
abstract = {Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 36,000 lncRNAs annotated in the human genome, we do not know the function of the majority. Here, we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important for viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 (RP11-542M13.2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP), and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways, while knockdown of OLMALINC impacted genes associated with the cell cycle. Data support both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Monocytes/cytology/metabolism
*Cell Proliferation
*CRISPR-Cas Systems
THP-1 Cells
Interferon Regulatory Factors/genetics/metabolism
Apoptosis

@article {pmid40193304,
year = {2025},
author = {Florindi, C and Jang, Y and Shani, K and Moretti, P and Bertarelli, C and Lanzani, G and Parker, KK and Lodola, F and Vurro, V},
title = {A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {217},
pages = {},
doi = {10.3791/67823},
pmid = {40193304},
issn = {1940-087X},
mesh = {*Photic Stimulation ; *Light Signal Transduction ; *Calcium/metabolism ; Induced Pluripotent Stem Cells ; Humans ; *Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9 ; Cations, Divalent/metabolism ; },
abstract = {In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca[2+] dye (X-Rhod 1) for monitoring the system's response. The results demonstrate that Ziapin2 can photomodulate Ca[2+] responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.},
}

*Photic Stimulation
*Light Signal Transduction
*Calcium/metabolism
Induced Pluripotent Stem Cells
Humans
*Myocytes, Cardiac/metabolism
CRISPR-Cas Systems
CRISPR-Associated Protein 9
Cations, Divalent/metabolism

@article {pmid40624545,
year = {2025},
author = {Ahmed, HMM and Zheng, L and Hunnekuhl, VS},
title = {Transferable approaches to CRISPR-Cas9 induced genome editing in non-model insects: a brief guide.},
journal = {Frontiers in zoology},
volume = {22},
number = {1},
pages = {13},
pmid = {40624545},
issn = {1742-9994},
support = {TE1380/1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Despite the large variety of insect species with divergent morphological, developmental and physiological features questions on gene function could for a long time only be addressed in few model species. The adaption of the bacterial CRISPR-Cas system for genome editing in eukaryotic cells widened the scope of the field of functional genetics: for the first time the creation of heritable genetic changes had become possible in a very broad range of organisms. Since then, targeted genome editing using the CRISPR-Cas technology has greatly increased the possibilities for genetic manipulation in non-model insects where molecular genetic tools were little established. The technology allows for site-specific mutagenesis and germline transformation. Importantly, it can be used for the generation of gene knock-outs, and for the knock-in of transgenes and generation of gene-reporter fusions. CRISPR-Cas induced genome editing can thus be applied to address questions in basic research in various insect species and other study organisms. Notably, it can also be used in applied insect biotechnology to design new pest and vector control strategies such as gene drives and precision guided Sterile Insect Technique. However, establishing CRISPR in a new model requires several practical considerations that depend on the scientific questions and on the characteristics of the respective study organism. Therefore, this review is intended to give a literature overview on different CRISPR-Cas9 based methods that have already been established in diverse insects. After discussing some required pre-conditions of the study organism, we provide a guide through experimental considerations when planning to conduct CRISPR-Cas9 genome editing, such as the design and delivery of guide RNAs, and of Cas9 endonuclease. We discuss the use of different repair mechanisms including homology directed repair (HDR) for a defined insertion of genetic elements. Furthermore, we describe different molecular methods for genetic screening and the use of visible markers. We focus our review on experimental work in insects, but due to the ubiquitous functionality of the CRISPR-Cas system many considerations are transferable to other non-model organisms.},
}

@article {pmid40623912,
year = {2025},
author = {Wang, YW and Tang, YM},
title = {[Advances in the application strategies of CRISPR/Cas9 technology in chimeric antigen receptor T cell therapy for hematological malignancies].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {46},
number = {5},
pages = {481-488},
doi = {10.3760/cma.j.cn121090-20240911-00343},
pmid = {40623912},
issn = {0253-2727},
mesh = {Humans ; *CRISPR-Cas Systems ; *Hematologic Neoplasms/therapy ; *Receptors, Chimeric Antigen ; *Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Gene Editing ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy has achieved breakthroughs in treating relapsed/refractory B-cell malignancies. However, it still faces challenges, including complex manufacturing processes, limited indications, T-cell exhaustion, and insufficient durability of therapeutic efficacy. CRISPR/Cas9, a highly efficient and relatively simple gene-editing technology, offers new avenues for overcoming these limitations. This review briefly outlines the working mechanism of CRISPR/Cas9 and focuses on its recent applications and clinical practices in developing universal CAR T-cells, enhancing T-cell function, and extending CAR T-cell therapy to T-cell and myeloid leukemias. Furthermore, this review highlights optimization strategies developed over the past two years to enhance the editing precision, delivery efficiency, and safety of the CRISPR/Cas9 system, aiming to provide insights for the optimal design and clinical application of CAR T-cell therapy.},
}

Humans
*CRISPR-Cas Systems
*Hematologic Neoplasms/therapy
*Receptors, Chimeric Antigen
*Immunotherapy, Adoptive/methods
T-Lymphocytes/immunology
Gene Editing

@article {pmid40623181,
year = {2025},
author = {Aumann, RA and Gouvi, G and Gregoriou, ME and Rehling, T and Sollazzo, G and Bourtzis, K and Schetelig, MF},
title = {Decoding and engineering temperature-sensitive lethality in Ceratitis capitata for pest control.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {28},
pages = {e2503604122},
doi = {10.1073/pnas.2503604122},
pmid = {40623181},
issn = {1091-6490},
support = {FF17000//SITplus, Hort Innovation/ ; SCHE 1833/7-1//Deutsche Forschungsgemeinschaft (DFG)/ ; 101059523//Horizon Europe Research and Innovation Action/ ; },
mesh = {Animals ; *Ceratitis capitata/genetics ; *Pest Control, Biological/methods ; Male ; Female ; Temperature ; Mutation ; Phenotype ; },
abstract = {The Sterile Insect Technique (SIT) is a species-specific and environmentally friendly method for effectively controlling pest insect populations based on releasing reared, sterile insects into infested areas. Sex sorting in rearing facilities, enabling male-only releases, is necessary to ensure SIT programs are efficient, cost-effective and, in case of mosquito control, also safe. This can be greatly facilitated by genetic sexing strains (GSS), exhibiting sex-specific phenotypic markers. However, the development of GSS remains challenging. The construction of a temperature-sensitive lethal (tsl)-based GSS in the Mediterranean fruit fly (Ceratitis capitata) over three decades ago was considered a major breakthrough for SIT programs but was never successfully replicated in other pests. After over 30 y of research, we have pinpointed a specific mutation in the C. capitata lysine--tRNA ligase (Lysyl-tRNA synthetase, LysRS) gene responsible for the tsl phenotype. Introducing this specific mutation into a wild-type strain produced full embryonic lethality under heat stress, replicating the original mutant phenotype. The random integration of a LysRS minigene reversed this effect. The high conservation of LysRS among insects suggests that tsl-based GSS could be expanded to multiple pest species and extend applications of SIT programs for disease prevention and the protection of agriculture.},
}

Animals
*Ceratitis capitata/genetics
*Pest Control, Biological/methods
Male
Female
Temperature
Mutation
Phenotype

@article {pmid40622878,
year = {2025},
author = {Perry, JK and Schwartzberg, PL and Golec, DP},
title = {Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {220},
pages = {},
doi = {10.3791/67380},
pmid = {40622878},
issn = {1940-087X},
mesh = {Animals ; Mice ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems ; *CD4-Positive T-Lymphocytes/cytology/immunology ; Cell Differentiation/genetics ; *Gene Knockout Techniques/methods ; Mice, Knockout ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies. Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.},
}

Animals
Mice
*Ribonucleoproteins/genetics
*CRISPR-Cas Systems
*CD4-Positive T-Lymphocytes/cytology/immunology
Cell Differentiation/genetics
*Gene Knockout Techniques/methods
Mice, Knockout
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40622492,
year = {2025},
author = {Parmar, H and Goel, A and Gelaw, TA and Reddy, MK},
title = {Enhancing drought resilience in crops: mechanistic approaches in the face of climate challenge.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {82},
pmid = {40622492},
issn = {1573-5028},
support = {09/512(0244)/2019-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; },
mesh = {*Crops, Agricultural/genetics/physiology ; *Droughts ; Climate Change ; Quantitative Trait Loci/genetics ; Stress, Physiological ; Plant Breeding ; Gene Editing ; Gene Expression Regulation, Plant ; },
abstract = {Enhancing drought resilience in crops has become a critical challenge in the face of global climate change, which is exacerbating the frequency and severity of drought events. This review explores mechanistic approaches aimed to improve crop drought tolerance, focusing on physiological, biochemical, and molecular mechanisms. We examine the key molecular pathways involved in drought stress responses, including the Mitogen-Activated Protein Kinase (MAPKs) signaling pathway, hormonal regulation, transcriptional control, and post-translational modifications such as ubiquitination-mediated protein degradation, and plant-microbe interaction. The review also delves into the mechanisms of drought stress tolerance, including drought escape, avoidance, and tolerance. It highlights significant traits contributing to drought resilience, such as stomatal regulation and root architecture. Furthermore, we discuss genomics and breeding approaches, including quantitative trait loci (QTL) mapping, marker-assisted selection (MAS), and cutting-edge CRISPR-Cas-based genome editing technologies. These advanced techniques, such as base editing, prime editing, and multiplexing, transform crop improvement strategies by facilitating precise and efficient modifications for enhanced drought resilience, with the success stories in crops such as rice, maize, wheat, and others. Integrating these mechanistic and technological approaches offers promising avenues for developing drought-resilient crops, ensuring food security under increasingly unpredictable climate conditions.},
}

*Crops, Agricultural/genetics/physiology
*Droughts
Climate Change
Quantitative Trait Loci/genetics
Stress, Physiological
Plant Breeding
Gene Editing
Gene Expression Regulation, Plant

@article {pmid40619763,
year = {2025},
author = {Fritz, B and Lapp, CJ and Gescher, J},
title = {Influence of Different Transposon Families on Genomic Stability of Shewanella oneidensis MR1.},
journal = {Microbial biotechnology},
volume = {18},
number = {7},
pages = {e70188},
doi = {10.1111/1751-7915.70188},
pmid = {40619763},
issn = {1751-7915},
support = {031B0847A//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Shewanella/genetics ; *DNA Transposable Elements/genetics ; *Genomic Instability ; Transposases/genetics/metabolism ; Genome, Bacterial ; Plasmids/genetics ; Whole Genome Sequencing ; },
abstract = {Shewanella oneidensis, recognised as an important model organism for exoelectrogenic electron transport, has been extensively studied for its potential applications in bioelectrochemical systems. To date, the activity of transposable elements in this organism has not been conclusively investigated. This study focused on transposases, specifically insertion sequences (IS), which make up approximately 4.7% of the organism's genome, and evaluated their impact on genome stability under stress conditions. Using whole genome sequencing, two IS families, ISSOD1 and ISSOD2, were identified as the most active, both showing similar transposition patterns across all tested stressors. A CRISPR/dCas9 cytosine deaminase system was used to introduce stop codons in the ISSOD2 transposase genes, resulting in a significant reduction of transposition events under stress conditions. Analysis of transposition patterns revealed a high frequency of insertions occurring on the megaplasmid, which predominantly carries non-essential genes. Experiments performed here to delete the megaplasmid resulted in the elimination of approximately 35% of its sequence, including an unexpected complete loss of the ori/repA region. Therefore, it was hypothesised that the megaplasmid either exists in a metastable state, possibly representing a cointegrated intermediate within the ISSOD9 (Tn3 member) transposition mechanism, or consists of two replicons that have been combined in previous assemblies due to long overlapping homologies resulting from the presence of ISSOD9. These findings highlight the dynamics of transposable elements in S. oneidensis and suggest strategies to improve strain stability by inactivating these elements and at least reducing megaplasmid sequences. Such approaches could improve the suitability of the organism for industrial applications.},
}

*Shewanella/genetics
*DNA Transposable Elements/genetics
*Genomic Instability
Transposases/genetics/metabolism
Genome, Bacterial
Plasmids/genetics
Whole Genome Sequencing

@article {pmid40384567,
year = {2025},
author = {Schneider, PG and Liu, S and Bullinger, L and Ostendorf, BN},
title = {BEscreen: a versatile toolkit to design base editing libraries.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W68-W72},
doi = {10.1093/nar/gkaf406},
pmid = {40384567},
issn = {1362-4962},
support = {70114327//German Cancer Aid/ ; 10.24.1.010MN//Fritz Thyssen/ ; //Guangzhou/ ; //Charité-BIH Digital Clinician/ ; //German Research Foundation/ ; },
mesh = {*Software ; *Gene Editing/methods ; *Gene Library ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Internet ; CRISPR-Cas Systems ; },
abstract = {Base editing enables the high-throughput screening of genetic variants for phenotypic effects. Base editing screens require the design of single guide RNA (sgRNA) libraries to enable either gene- or variant-centric approaches. While computational tools supporting the design of sgRNAs exist, no solution offers versatile and scalable library design enabling all major use cases. Here, we introduce BEscreen, a comprehensive base editing guide design tool provided as a web server (bescreen.ostendorflab.org) and as a command line tool. BEscreen provides variant-, gene-, and region-centric modes to accommodate various screening approaches. The variant mode accepts genomic coordinates, amino acid changes, or rsIDs as input. The gene mode designs near-saturation libraries covering the entire coding sequence of given genes or transcripts, and the region mode designs all possible guides for given genomic regions. BEscreen enables selection of guides by biological consequence, it features comprehensive customization of base editor characteristics, and it offers optional annotation using Ensembl's Variant Effect Predictor. In sum, BEscreen is a highly versatile tool to design base editing screens for a wide range of use cases with seamless scalability from individual variants to large, near-saturation libraries.},
}

*Software
*Gene Editing/methods
*Gene Library
*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Internet
CRISPR-Cas Systems

@article {pmid40377102,
year = {2025},
author = {Chapdelaine-Trépanier, V and Shenoy, S and Masud, W and Minju-Op, A and Bérubé, MA and Schönherr, S and Forer, L and Fradet-Turcotte, A and Taliun, D and Cuella-Martin, R},
title = {CRISPR-BEasy: a free web-based service for designing sgRNA tiling libraries for CRISPR-dependent base editing screens.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W193-W202},
doi = {10.1093/nar/gkaf382},
pmid = {40377102},
issn = {1362-4962},
support = {FRN:DV2-197674/CAPMC/CIHR/Canada ; //Canada Excellence Chair in Genomic Medicine/ ; //McGill University/ ; //Fonds de Recherche du Québec-Santé/ ; //Doctoral Training Scholarships/ ; //Canada Research Chair Tier II in Molecular Biology and Genomic Instability/ ; FRN:DV2-197674/CAPMC/CIHR/Canada ; },
mesh = {Internet ; *Gene Editing/methods ; *Software ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Humans ; },
abstract = {CRISPR-dependent base editing (BE) enables the modeling and correction of genetic mutations at single-base resolution. Base editing screens, where point mutations are queried en masse, are powerful tools to systematically draw genotype-phenotype associations and characterise the function of genes and other genomic elements. However, the lack of user-friendly web-based tools for designing base editing screens can hinder broad technology adoption. Here, we introduce CRISPR-BEasy (https://crispr-beasy.cerc-genomic-medicine.ca), a free, automated web-based server that streamlines the creation of single guide (sg)RNA tiling libraries for base editing screens. Researchers can provide their genes or genomic features of interest, their base editors of choice, and target sequences to act as positive and negative controls. The server designs and annotates sgRNA libraries by integrating custom code with publicly available tools such as crisprVerse and Ensembl's Variant Effect Predictor. CRISPR-BEasy provides downloadable results, including sgRNA on/off-target scores, predicted mutational outcomes per base editor, and intuitive interactive visualizations for data quality assessment. CRISPR-BEasy also provides a separate tool that assembles sgRNA libraries into oligonucleotides for cloning following the detailed protocol documented in the searchable web server manual. Together, CRISPR-BEasy ensures the seamless design of cloning-ready sgRNA libraries, seeking to democratise access to base editing screening technologies.},
}

Internet
*Gene Editing/methods
*Software
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
Humans

@article {pmid40337925,
year = {2025},
author = {Mekonnen, AM and Seong, K and Kim, H and Park, J},
title = {Variant-aware Cas-OFFinder: web-based in silico variant-aware potential off-target site identification for genome editing applications.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W118-W124},
doi = {10.1093/nar/gkaf389},
pmid = {40337925},
issn = {1362-4962},
support = {RS-2024-00322053//Rural Development Administration/ ; //National Research Foundation of Korea/ ; //Korean government/ ; RS-2023-00278658//MSIT/ ; //Bio & Medical Technology Development/ ; RS-2024-00439078//Korean government (MSIT)/ ; BK21 Four//Korean government (MSIT)/ ; },
mesh = {*Gene Editing/methods ; *Software ; Humans ; Internet ; *CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; Haplotypes ; Computer Simulation ; },
abstract = {Genome editing based on CRISPR systems has been widely used in the vast areas of biomedical and agricultural applications. However, identifying the potential off-target sites remains challenging, particularly in individuals with diverse genetic variations. Several in silico tools have been developed to predict potential off-target sites, but they have limitations on their performance and scalability. In this paper, we present "Variant-aware Cas-OFFinder," a novel pipeline based on Cas-OFFinder for identifying potential off-target sites by accounting for individual genetic variants. We benchmarked the pipeline's improved scalability and performance with the human genome and pepper cultivars, having unique potential off-target sites on each allele at the haplotype level. The web tool is open to all users without a login requirement and is freely available online at https://rgetoolkit.com/var-cas-offinder.},
}

*Gene Editing/methods
*Software
Humans
Internet
*CRISPR-Cas Systems
Genome, Human
Genetic Variation
Haplotypes
Computer Simulation

@article {pmid40334276,
year = {2025},
author = {Liu, H and Dong, J and Wu, R and Dai, J and Lou, X and Xia, F and Willner, I and Huang, F},
title = {Light-Triggered CRISPR/Cas12a for Genomic Editing and Tumor Regression.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {28},
pages = {e202502892},
doi = {10.1002/anie.202502892},
pmid = {40334276},
issn = {1521-3773},
support = {22377112//National Natural Science Foundation of China/ ; 22090050//National Natural Science Foundation of China/ ; 2021YFA1200400//National Key R&D Program of China/ ; 22161142020//Joint NSFC-ISF Research Grant Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Light ; Animals ; Hep G2 Cells ; Mice ; Hepatocyte Growth Factor/genetics ; Cell Proliferation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A photo-triggered CRISPR/Cas12a machinery for in vitro and in vivo gene editing is introduced. The system consists of a caged, inactive ortho-nitrobenzyl phosphate ester photo-responsive crRNA, which, upon light-induced deprotection, yields the active CRISPR/Cas12a gene editing machinery (LAC12aGE). The LAC12aGE system induces specific thymidine-rich (TTTN) protospacer-adjacent motif (PAM)-guided double-stranded breaks in genomic DNA, which upon non-homologous end-joining lead to gene repair. The LAC12aGE machinery is applied for gene editing of an exogenous dual fluorescent reporter gene in living cells, as well as the endogenous gene encoding DNA methyltransferase 1. In addition, the LAC12aGE is applied for in vitro gene editing and disruption of the hepatocyte growth factor (HGF) gene in HepG2 cells, where knockout of the HGF gene results in inhibited cell proliferation and migration, as well as enhanced apoptosis. Moreover, the in vivo knockout and disruption of the HGF gene in HepG2 tumors by the LAC12aGE machinery is demonstrated. The cyclic temporal development of the LAC12aGE system in tumors shows effective inhibition of tumor growth and enhanced apoptosis/necrosis of tumor tissues compared to control systems.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*Light
Animals
Hep G2 Cells
Mice
Hepatocyte Growth Factor/genetics
Cell Proliferation
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40308216,
year = {2025},
author = {Marino, GB and Evangelista, JE and Clarke, DJB and Ma'ayan, A},
title = {L2S2: chemical perturbation and CRISPR KO LINCS L1000 signature search engine.},
journal = {Nucleic acids research},
volume = {53},
number = {W1},
pages = {W338-W350},
doi = {10.1093/nar/gkaf373},
pmid = {40308216},
issn = {1362-4962},
support = {OT2OD036435/GF/NIH HHS/United States ; OT2OD030160/GF/NIH HHS/United States ; U24CA264250/GF/NIH HHS/United States ; U24CA271114/GF/NIH HHS/United States ; R01DK131525/GF/NIH HHS/United States ; RC2DK131995/GF/NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; *Software ; Internet ; *Search Engine ; Gene Expression Profiling ; Small Molecule Libraries/pharmacology ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptome ; },
abstract = {As part of the Library of Integrated Network-Based Cellular Signatures (LINCS) NIH initiative, 248 human cell lines were profiled with the L1000 assay to measure the effect of 33 621 small molecules and 7508 single-gene CRISPR knockouts. From this massive dataset, we computed 1.678 million sets of up- and down-regulated genes. These gene sets are served for search by the LINCS L1000 Signature Search (L2S2) web server application. With L2S2, users can identify small molecules and single gene CRISPR KOs that produce gene expression profiles similar or opposite to their submitted single or up/down gene sets. L2S2 also includes a consensus search feature that ranks perturbations across all cellular contexts, time points, and concentrations. To demonstrate the utility of L2S2, we crossed the L2S2 gene sets with gene sets collected for the RummaGEO resource. The analysis identified clusters of differentially expressed genes that match drug classes, tissues, and diseases, pointing to many opportunities for drug repurposing and drug discovery. Overall, the L2S2 web server application can be used to further the development of personalized therapeutics while expanding our understanding of complex human diseases. The L2S2 web server application is available at https://l2s2.maayanlab.cloud.},
}

Humans
*CRISPR-Cas Systems
Gene Knockout Techniques
*Software
Internet
*Search Engine
Gene Expression Profiling
Small Molecule Libraries/pharmacology
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats
Transcriptome

@article {pmid39729054,
year = {2025},
author = {Ballantine, J and Tisdale, JF},
title = {Gene therapy for sickle cell disease: recent advances, clinical trials and future directions.},
journal = {Cytotherapy},
volume = {27},
number = {7},
pages = {826-834},
pmid = {39729054},
issn = {1477-2566},
support = {T32 CA060441/CA/NCI NIH HHS/United States ; ZIA HL006008/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods ; Gene Editing ; Clinical Trials as Topic ; Hematopoietic Stem Cell Transplantation ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Animals ; beta-Thalassemia/therapy/genetics ; },
abstract = {Sickle cell disease (SCD) is the most common inherited blood disorder worldwide, impacting millions and imposing severe healthcare challenges, particularly in resource-limited regions. Current treatments have variable efficacy and require lifelong adherence. Allogeneic Hematopoietic Stem Cell Transplantation can be curative but comes with significant side effects and limited donor availability limits its widespread applicability. Gene therapy, by addressing the root genetic causes, offers a revolutionary alternative. This article discusses the molecular mechanisms of SCD and β-thalassemia and highlights advancements in gene therapy, such as gene addition via lentiviral vectors and gene editing with CRISPR/Cas9 technology. Clinical trials have brought about significant progress but challenges remain, including leukemogenesis, delivery efficiency and cost. Future efforts must focus on enhancing efficiency, reducing costs, developing nongenotoxic conditioning regimens and methods for in vivo application.},
}

Humans
*Anemia, Sickle Cell/therapy/genetics
*Genetic Therapy/methods
Gene Editing
Clinical Trials as Topic
Hematopoietic Stem Cell Transplantation
CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
Animals
beta-Thalassemia/therapy/genetics

@article {pmid40619482,
year = {2025},
author = {Bodmer, N and Uth, K and Mehmeti, R and Demir, CS and Stroka, D and Ghaffari-Tabrizi-Wizsy, N and Lugli, A and de Wever, O and Zlobec, I and Tschan, MP},
title = {CDX2 loss in colorectal cancer cells is associated with invasive properties and tumor budding.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {24113},
pmid = {40619482},
issn = {2045-2322},
support = {ESKAS, nr. 2021.0019 / Kosovo / OP//Swiss Government Excellence Scholarship/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; 31003A_166578/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
mesh = {*CDX2 Transcription Factor/genetics/metabolism ; *Colorectal Neoplasms/pathology/genetics/metabolism ; Humans ; Animals ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; Mice ; Cell Proliferation ; Epithelial-Mesenchymal Transition/genetics ; Cadherins/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {In colorectal cancer (CRC), tumor buds (TB) are observed histologically as single tumor cell or small tumor cell clusters located mainly at the advancing tumor edge. TB are a marker of poor prognosis and correlate with metastatic disease in CRC patients. They often lack expression of CDX2 and overexpress markers involved in epithelial-mesenchymal transition (EMT). We evaluated the function of CDX2 in CRC proliferation and migration using CRISPR/Cas9 technology and demonstrated a possible link to tumor dissociation and tumor budding. Knocking out CDX2 in CRC cell lines significantly increased migration. Importantly, the observed phenotypes could be rescued by re-expressing CDX2 and by specific CRISPR synergistic activation mediator (SAM) of endogenous CDX2 in CDX2 low expressing CRC cell lines. Multiplex immunofluorescence (mIF) analysis of primary tumor regions compared to TB in a CDX2-positive CRC patient sample as well as patient derived xenografts (PDX) revealed significantly lower CDX2 expression and correlating E-cadherin levels in TB compared to primary tumor regions, in both models. Accordingly, increased invasiveness of CRC CDX2 knockout cells was seen in ex ovo xenografts. Taken together, our results provide further insight into the function of CDX2 in preventing CRC cell migration, tumor budding and tumor aggressiveness.},
}

*CDX2 Transcription Factor/genetics/metabolism
*Colorectal Neoplasms/pathology/genetics/metabolism
Humans
Animals
Cell Movement/genetics
Cell Line, Tumor
Neoplasm Invasiveness
Mice
Cell Proliferation
Epithelial-Mesenchymal Transition/genetics
Cadherins/metabolism
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems

@article {pmid40617436,
year = {2025},
author = {Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Structure-transformable poly (thymine) activators of CRISPR/Cas12a for highly sensitive detection of mercury (II) ions.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 4},
pages = {145748},
doi = {10.1016/j.ijbiomac.2025.145748},
pmid = {40617436},
issn = {1879-0003},
abstract = {Off-target effects of the CRISPR/Cas system refer to suboptimal activity triggered by activators containing unintended modifications beyond the intended target, which can lead to unwanted genetic changes-particularly problematic in therapeutic applications. In this study, the off-target effects of Cas12a are strategically repurposed for advanced Hg[2+] detection by leveraging a structure-transformable activator. The proposed approach integrates a two-segment poly-T DNA activator with the CRISPR/Cas12a system, where the activator forms hairpin structures through thymine-Hg[2+]-thymine base pairing in response to mercury concentrations. Increasing Hg[2+] concentrations promote the formation and stability of hairpin DNA, significantly suppressing Cas12a activity and resulting in an ON-OFF fluorescence signal modulated by trans-cleavage activity. The effect of varying thymine segment lengths in the poly-T activator was investigated to increase the hybridization burden for crRNA, ultimately enhancing detection sensitivity. The Hg[2+]-dependent structural transformation of the activator was characterized using circular dichroism (CD), while urea and native PAGE analyses confirmed the catalytic behavior of both cis- and trans-cleavage activities, respectively, as a function of Hg[2+] concentration. The trans-cleavage efficiency (kcat/KM) decreased by 3.03-fold as Hg[2+] concentration increased from 0 to 40 nM. Under optimized conditions, the biosensor demonstrated a linear detection range of 0-30 nM, a detection limit as low as 0.372 nM, and high selectivity for Hg[2+] over other metal ions. Furthermore, the biosensor was successfully applied for Hg[2+] detection in field water samples. These findings establish a robust foundation for exploiting the off-target effects of the CRISPR/Cas12a system for the efficient detection of heavy metals in environmental monitoring.},
}

@article {pmid40617152,
year = {2025},
author = {Rangu, SS and Misra, CS and Shaikh, S and Singh, M and Bindal, G and Singh, S and Purageri, S and Shettigar, S and Warke, R and Cherian, S and Rath, D},
title = {RNA extraction-free CRISPR-based SARS-CoV-2 detection in viral transport medium and dry swab-a comparative analysis: Short title: Extraction-free CRISPR-based Covid detection.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {3},
pages = {116982},
doi = {10.1016/j.diagmicrobio.2025.116982},
pmid = {40617152},
issn = {1879-0070},
abstract = {CRISPR-Cas-based methods have shown high efficacy in detecting SARS-CoV-2. Amidst the Covid-19 pandemic, numerous studies have explored SARS-CoV-2 detection methods without the need for RNA extraction, aiming to reduce cost and processing time. Here, we assessed a CRISPR-based SARS-CoV-2 detection method's ability to detect the virus in viral transport medium (VTM). Swabs obtained from Covid-19 positive patients and stored in two Indian brands of VTM were examined alongside dry swab samples. The samples underwent proteinase -K treatment followed by heat incubation. The released nucleic acids were tested by RT-LAMP and CRISPR-based detection. We conclude that SARS-CoV-2 can be detected in VTM of two commercial preparations as well as dry swab samples without RNA extraction and purification. COVID detection was found to be more efficient for dry swab samples compared to VTM samples. The work flow described in this paper can be extended to other respiratory diseases.},
}

@article {pmid40057303,
year = {2025},
author = {Wang, L and Hu, Y and Qiu, Y and Lin, H and Li, X and Fu, S and Zeng, YY and Ghouse, M and Long, C and Liu, Y and Fei, JF},
title = {Establishing a semi-homology-directed recombination method for precision gene integration in axolotls.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {52},
number = {7},
pages = {942-953},
doi = {10.1016/j.jgg.2025.03.001},
pmid = {40057303},
issn = {1673-8527},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Ambystoma mexicanum/genetics ; Gene Editing/methods ; SOXB1 Transcription Factors/genetics ; *Homologous Recombination ; },
abstract = {The axolotl is broadly used in regenerative, developmental, and evolutionary biology research. Targeted gene knock-in is crucial for precision transgenesis, enabling disease modeling, visualization, tracking, and functional manipulation of specific cells or genes of interest (GOIs). Existing CRISPR/Cas9-mediated homology-independent method for gene knock-in often causes "scars/indels" at integration junctions. Here, we develop a CRISPR/Cas9-mediated semi-homology-directed recombination (HDR) knock-in method using a donor construct containing a single homology arm for the precise integration of GOIs. This semi-HDR approach achieves seamless single-end integration of the Cherry reporter gene and a large inducible Cre cassette into intronless genes like Sox2 and Neurod6 in axolotls, which are challenging to modify with the homology-independent method. Additionally, we integrate the inducible Cre cassette into intron-containing loci (e.g., Nkx2.2 and FoxA2) without introducing indels via semi-HDR. GOIs are properly expressed in F0 founders, with approximately 5%-10% showing precise integration confirmed by genotyping. Furthermore, using the Nkx2.2:CreER[T2] line, we fate-map spinal cord p3 neural progenitor cells, revealing that Nkx2.2[+] cells adopt different lineages in development and regeneration, preferentially generating motoneurons over oligodendrocytes during regeneration. Overall, this semi-HDR method balances efficiency and precision in the integration of GOIs, providing a valuable tool for generating knock-in axolotls and potentially extending to other species.},
}

Animals
CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Ambystoma mexicanum/genetics
Gene Editing/methods
SOXB1 Transcription Factors/genetics
*Homologous Recombination

@article {pmid40616579,
year = {2025},
author = {Urnov, F and Kassim, S and Musunuru, K and Liu, D and Lee, A and Barrera, L and Stetkiewicz, P and Bruno, J and Hewitt, M and Lister, T and Malech, H and Gasch, L and Diver, M and Gertler, N and Grignon, F and Le, A and Lehmicke, M and Almendro-Navarro, V and Lembong, J},
title = {Advancing gene-editing platforms to improve the viability of rare-disease therapeutics: key insights from a 2024 Scientific Exchange hosted by ARM, ISCT, and Danaher.},
journal = {Cytotherapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jcyt.2025.06.010},
pmid = {40616579},
issn = {1477-2566},
abstract = {Rare-disease therapeutics face viability challenges due to small patient populations and drug-development and regulatory frameworks that were not developed to address rapidly progressive or quickly fatal conditions. Because the majority of rare diseases are genetic in nature, gene-editing modalities offer substantial promise. This Scientific Exchange, co-hosted by the Alliance for Regenerative Medicine, the International Society for Cell and Gene Therapy, and Danaher Corporation in November 2024, set out to address the challenge of realizing the full promise of gene editing for rare-disease therapies by advancing platforms that leverage stable and reusable processes or components to develop multiple therapies. Through multi-stakeholder engagement and discussions of case studies in CRISPR/Cas nuclease, base, and prime editing, 4 key opportunities emerged that deliver value by holding platform elements constant and/or streamlining development steps: (1) consistent delivery vehicle; (2) consistent manufacturing; (3) benefit-risk appropriate quality requirements; and (4) expansive clinical trial designs. Together, these opportunities could yield up to 5-fold efficiency gains and result in substantial value creation for patients, regulators, and developers, potentially decreasing the time required to dose patients with a new gene-editing therapy from years down to 6 months.},
}

@article {pmid40615444,
year = {2025},
author = {Novotná, M and Tinti, M and Faria, JRC and Horn, D},
title = {Precision-edited histone tails disrupt polycistronic gene expression controls in trypanosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6194},
pmid = {40615444},
issn = {2041-1723},
support = {217105/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {*Histones/genetics/metabolism ; *Trypanosoma brucei brucei/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; RNA Polymerase II/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems ; *Gene Expression Regulation ; Gene Editing ; Mutation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Methylation ; Gene Expression Profiling ; Chromatin/metabolism ; },
abstract = {Transcription of protein coding genes in trypanosomatids is atypical and almost exclusively polycistronic. In Trypanosoma brucei, for example, approximately 150 polycistrons, and 8000 genes, are constitutively transcribed by RNA polymerase II. The RNA pol-II promoters are also unconventional and characterised by regions of chromatin enriched for histones with specific patterns of post-translational modification on their divergent N-terminal tails. To investigate the roles of histone tail-residues in gene expression control in T. brucei, we engineered strains exclusively expressing mutant histones. We used an inducible CRISPR-Cas9 system to delete >40 histone H4 genes, complementing the defect with a single ectopic H4 gene. The resulting "hist[one]H4" strains were validated using whole-genome sequencing and transcriptome analysis. We then performed saturation mutagenesis of six histone H4 N-terminal tail lysine residues, that are either acetylated or methylated, and profiled relative fitness of 384 distinct precision-edited mutants. H4[lys10] mutations were not tolerated, but we derived nineteen strains exclusively expressing distinct H4[lys4] or H4[lys14] mutants. Proteomic and transcriptomic analysis of H4[lys4] glutamine mutants revealed significantly reduced expression of genes adjacent to RNA pol-II promoters, where glutamine mimics abnormally elevated acetylation. Thus, we present direct evidence for polycistronic expression control by modified histone H4 N-terminal tail residues in trypanosomes.},
}

*Histones/genetics/metabolism
*Trypanosoma brucei brucei/genetics/metabolism
Protozoan Proteins/genetics/metabolism
RNA Polymerase II/metabolism/genetics
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems
*Gene Expression Regulation
Gene Editing
Mutation
Acetylation
Lysine/metabolism
Protein Processing, Post-Translational
Methylation
Gene Expression Profiling
Chromatin/metabolism

@article {pmid40615387,
year = {2025},
author = {Lou, S and DJiake Tihagam, R and Wasko, UN and Equbal, Z and Venkatesan, S and Braczyk, K and Przanowski, P and Il Koo, B and Saltani, I and Singh, AT and Likhite, S and Powers, S and Souza, GMPR and Maxwell, RA and Yu, J and Zhu, LJ and Beenhakker, M and Abbott, SBG and Lu, Z and Green, MR and Meyer, KC and Tushir-Singh, J and Bhatnagar, S},
title = {Targeting microRNA-dependent control of X chromosome inactivation improves the Rett Syndrome phenotype.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6169},
pmid = {40615387},
issn = {2041-1723},
mesh = {*Rett Syndrome/genetics/therapy ; *MicroRNAs/genetics/metabolism ; *X Chromosome Inactivation/genetics ; Animals ; Female ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Humans ; Mice ; RNA, Long Noncoding/genetics/metabolism ; Phenotype ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; Male ; },
abstract = {X chromosome inactivation (XCI) is induced by Xist long non-coding RNA and protein-coding genes. However, the role of small non-coding RNA function in XCI remains unidentified. Our genome-wide, loss-of-function CRISPR/Cas9 screen in female fibroblasts identified microRNAs (miRNAs) as regulators of XCI. A striking finding is the identification of miR106a among the top candidates from the screen. Loss of miR106a is accompanied by altered Xist interactome, leading to dissociation and destabilization of Xist. XCI interference via miR106a inhibition has therapeutic implications for Rett syndrome (RTT) girls with a defective X-linked MECP2 gene. Here, we discovered that the inhibition of miR106a significantly improves several facets of RTT pathology: it increases the life span, enhances locomotor activity and exploratory behavior, and diminishes breathing variabilities. Our results suggest that miR106a targeting offers a feasible therapeutic strategy for RTT and other monogenic X-linked neurodevelopmental disorders.},
}

*Rett Syndrome/genetics/therapy
*MicroRNAs/genetics/metabolism
*X Chromosome Inactivation/genetics
Animals
Female
Methyl-CpG-Binding Protein 2/genetics/metabolism
Humans
Mice
RNA, Long Noncoding/genetics/metabolism
Phenotype
Fibroblasts/metabolism
CRISPR-Cas Systems
Disease Models, Animal
Male

@article {pmid40613815,
year = {2025},
author = {Pinto, D and Mendes, I and Cunha, MV},
title = {Genomic Survey Reveals no Detectable Bacteriophage Activity in Mycobacterium bovis Across a Large Population.},
journal = {FEMS microbiology ecology},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsec/fiaf072},
pmid = {40613815},
issn = {1574-6941},
abstract = {Phages are major drivers of bacterial evolution, yet their ecological and evolutionary interactions with Mycobacterium bovis, a key member of the Mycobacterium tuberculosis complex (MTBC), remain understudied. In this work, we investigate the elusive phage-bacterium interface in M. bovis by integrating comparative genomics of 200 isolates from infected animals with molecular analyses of M. bovis-positive environmental samples. Despite employing diverse and complementary approaches, we found no evidence of active or recent phage infections: no novel prophages beyond the conserved phiRv1, no expansion of CRISPR arrays, and no co-occurrence of M. bovis and mycobacteriophages in host tissues or environmental matrices. Intriguingly, we identified multiple independent excision events of phiRv1 across closely related lineages, suggesting recent prophage mobilization driven by unidentified ecological or genomic triggers. These findings echo previous observations in M. tuberculosis and point toward a stable, phage-scarce landscape across MTBC members. Our results raise compelling questions about the barriers to phage predation in M. bovis, the functionality of its CRISPR-Cas system, and the selective pressures underlying prophage retention and loss. By shedding light on these underexplored dynamics, our study reveals critical gaps in the ecological understanding of M. bovis and highlights opportunities for phage-based innovation in TB control.},
}

@article {pmid40613714,
year = {2025},
author = {Wu, D and Snead, S and Ganguly, C and Babu, K and Li, Y and Kathiresan, V and Shen, R and Zhang, X and Rajan, R and Qin, PZ},
title = {Structural integrity and side-chain interaction at the loop region of the bridge helix modulate Cas9 substrate discrimination.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf557},
pmid = {40613714},
issn = {1362-4962},
support = {MCB-1716744//National Science Foundation/ ; MCB-1818107//National Science Foundation/ ; MCB-1716423//National Science Foundation/ ; MCB-2424888//National Science Foundation/ ; R35-GM145341/NH/NIH HHS/United States ; //Arnold and Mabel Beckman Foundation/ ; },
mesh = {*CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Substrate Specificity ; CRISPR-Cas Systems ; Models, Molecular ; Protein Binding ; DNA Cleavage ; Protein Conformation ; },
abstract = {CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) has been revolutionizing genome engineering, and in-depth understanding of mechanisms governing its DNA discrimination is critical for continuing technology advances. An arginine-rich bridge helix (BH) connecting the nuclease lobe and the recognition lobe, which is conserved across the Cas9 family, exists in a helix-loop-helix conformation in the apo wild-type protein but converts to a long contiguous helix in the Cas9/RNA binary complex. In this work, distances measured with spin labels were utilized to investigate BH's conformational transitions in the solution state upon single-guide RNA (sgRNA) binding, which is a critical early event preceding DNA binding and cleavage. Analyses show that sgRNA binding drives BH conformational changes in the wild-type SpyCas9 (SpyCas9WT) as well as in two BH-loop variants, SpyCas92Pro and SpyCas92Ala. Each Cas9-sgRNA binary complex, however, exhibits distinct BH features that reveal mutation-specific effects on helical integrity versus side-chain interactions. In addition, the BH conformational variations can be correlated to the observed changes in the mismatch cleavage profiles of the Cas9 variants. The work represents the first use of distances measured by site-directed spin labeling to investigate Cas9 protein conformational changes in the solution state and advances our understanding on the structure-dynamic-function relationship governing DNA target discrimination by Cas9.},
}

*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
DNA/metabolism/chemistry/genetics
Substrate Specificity
CRISPR-Cas Systems
Models, Molecular
Protein Binding
DNA Cleavage
Protein Conformation

@article {pmid40613710,
year = {2025},
author = {Hibshman, GN and Taylor, DW},
title = {Structural basis of a dual-function type II-B CRISPR-Cas9.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf585},
pmid = {40613710},
issn = {1362-4962},
support = {R35GM138348/NH/NIH HHS/United States ; F-1938//Welch Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; *Francisella/genetics/enzymology ; Gene Editing ; *Bacterial Proteins/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Streptococcus pyogenes/genetics/enzymology ; DNA/genetics/chemistry/metabolism ; DNA Cleavage ; },
abstract = {Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature-the REC3 clamp-that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a noncanonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved across type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the molecular basis of heightened specificity and virulence enabled by FnCas9, with broad implications for biotechnology and therapeutic development.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
*Francisella/genetics/enzymology
Gene Editing
*Bacterial Proteins/chemistry/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Streptococcus pyogenes/genetics/enzymology
DNA/genetics/chemistry/metabolism
DNA Cleavage

@article {pmid40613705,
year = {2025},
author = {Schwartz, CI and Abell, NS and Li, A and Aradhana, and Tycko, J and Truong, A and Montgomery, SB and Hess, GT},
title = {Toward optimizing diversifying base editors for high-throughput mutational scanning studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf620},
pmid = {40613705},
issn = {1362-4962},
support = {R35GM150462/NH/NIH HHS/United States ; NIH-1F99DK126120-01/NH/NIH HHS/United States ; NIH-4K00DK126120-03/NH/NIH HHS/United States ; S10RR025518-01/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Cytidine Deaminase/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; DNA Mutational Analysis/methods ; AICDA (Activation-Induced Cytidine Deaminase) ; },
abstract = {Base editors, including diversifying base editors that create C>N mutations, are potent tools for systematically installing point mutations in mammalian genomes and studying their effect on cellular function. Numerous base editor options are available for such studies, but little information exists on how the composition of the editor (deaminase, recruitment method, and fusion architecture) affects editing. To address this knowledge gap, the effect of various design features, such as deaminase recruitment and delivery method (electroporation or lentiviral transduction), on editing was assessed across ∼200 synthetic target sites. The direct fusion of a hyperactive variant of activation-induced cytidine deaminase to the N-terminus of dCas9 (DivA-BE) produced the highest editing efficiency, ∼4-fold better than the previous CRISPR-X method. Additionally, DivA-BE mutagenized the DNA strand that anneals to the targeting sgRNA (target strand) to create complementary C>N mutations, which were absent when the deaminase was fused to the C-terminus of dCas9. Based on these studies that comprehensively analyze the editing patterns of several popular base editors, DivA-BE editors efficiently diversified their target sites, albeit with increased indel frequencies. Overall, the improved editing efficiency makes the DivA-BE editors ideal for discovering functional variants in mutational scanning assays.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems
Cytidine Deaminase/genetics/metabolism
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Point Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Mutation
DNA Mutational Analysis/methods
AICDA (Activation-Induced Cytidine Deaminase)

@article {pmid40613203,
year = {2025},
author = {Criollo Delgado, L and Zamalutdinov, A and Potokina, E},
title = {Identification of Soybean E1-E4 Gene Orthologs in the Guar Genome Using Comprehensive Transcriptome Assembly and Annotation.},
journal = {Frontiers in bioscience (Scholar edition)},
volume = {17},
number = {2},
pages = {26548},
doi = {10.31083/FBS26548},
pmid = {40613203},
issn = {1945-0524},
support = {24-26-00073//Russian Science Foundation/ ; },
mesh = {*Glycine max/genetics ; *Cyamopsis/genetics ; *Transcriptome/genetics ; *Genome, Plant ; Molecular Sequence Annotation ; Photoperiod ; Gene Expression Regulation, Plant ; Plant Proteins/genetics ; *Genes, Plant ; Gene Expression Profiling ; Galactans ; Mannans ; Plant Gums ; },
abstract = {BACKGROUND: We publish the first available transcriptome assembly of guar (Cyamopsis tetragonoloba (L.) Taub.), a well-known source of guar gum (food additive E 412). At high latitudes, e.g., in Russia, the main challenge for guar cultivation is the long photoperiod during summer, which delays flowering and maturation of guar plants. Meanwhile, identifying of genes affecting the photoperiod sensitivity of guar would have a major impact on the development of marker-assisted breeding of this valuable food crop.

METHODS: RNA isolated from leaves of early and late flowering guar plants grown under long-day conditions were used to generate de novo transcriptome assembly. A similarity search was conducted using BLASTN 2.2.31+ with default settings to identify homologous sequences of soybean maturity genes E1-E4 in guar transcriptome and genome assembly. Gene prediction tools such as AUGUSTUS and FGENESH+ were used to predict the exon-intron structure of the candidate genes. Functional annotation of the amino acid sequence was performed using InterProScan v. 5.68-100.

RESULTS: The transcriptome assembly contained sequences of 96,447 clustered transcript isoforms in the leaves of guar plants grown under long-day conditions. The transcriptome assembly was annotated using BLAST against the Glycine max genome, and 42,615 guar transcripts (44.2%) were found to be similar to soybean genes. We used the developed transcriptome assembly to discover orthologs of the E1-E4 soybean loci in the guar genome that have the greatest impact on the flowering and maturation of this closely related, short-day legume crop. A high level of identity was detected between peptide sequences encoding by orthologous genes E1 and CtE1 (80%), E2 and CtE2 (93%), E3 and CtE3 (83%), and E4 and CtE4 (91%). The sequences and the intron-exon structure of the genes in soybean and guar were similar, suggesting that the genetic pathways underlying basic flowering mechanisms are conserved between these two legume crops.

CONCLUSIONS: The revealed intron-exon structure of the guar genes CtE1-CtE4 creates possibilities for their targeted mutagenesis, e.g., using CRISPR-Cas and developing new guar germplasm with low sensitivity to photoperiod.},
}

*Glycine max/genetics
*Cyamopsis/genetics
*Transcriptome/genetics
*Genome, Plant
Molecular Sequence Annotation
Photoperiod
Gene Expression Regulation, Plant
Plant Proteins/genetics
*Genes, Plant
Gene Expression Profiling
Galactans
Mannans
Plant Gums

@article {pmid40611477,
year = {2025},
author = {Bulle, M and Abbagani, S and Raza, A},
title = {Genome blaze: engineering chilli pepper chloroplasts for sustainable production of capsaicinoids through organellar genome editing.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {},
number = {},
pages = {},
doi = {10.1111/plb.70067},
pmid = {40611477},
issn = {1438-8677},
support = {PDF/2018/001226//Science and Engineering Research Board/ ; },
abstract = {The quest for superior parental lines of chilli pepper, enriched with heightened levels of phytochemicals, such as capsinoids (CATs) and capsaicinoids (CAPs), has recently gained momentum. Plant scientists now pay more attention to generating pepper cultivars that offer both increased yield and richness in these crucial ingredients. Indeed, the average pepper yield per hectare, as well as richness of these ingredients, are affected by multiple environmental stimuli. However, expression of specific genes is crucial to stimulate the CAT and CAP levels in response to environmental conditions. Recently, new technologies, like genome editing using CRISPR/Cas and engineering of chloroplasts, have been tested in chilli pepper. The advances in genome editing tools, such as the prime editor and base editor methods, have tremendous potential for plant organelle genome engineering. In this article, we highlight recent advancements in CRISPR/Cas and plastid engineering within Capsicum, coupled with application of base editing approaches for editing plant organelle DNA. We also assess the challenges and opportunities in the pursuit of commercial and sustainable production of bioactive compounds specific to Capsicum species.},
}

@article {pmid40611420,
year = {2025},
author = {Rezaei, M and Jalali, A and Sadah Al-Azzawi, DH},
title = {Engineered Bacteriophages: Advances in Phage Genome Redesign Strategies for Therapeutic and Environmental Applications.},
journal = {Protein and peptide letters},
volume = {},
number = {},
pages = {},
doi = {10.2174/0109298665372719250616085616},
pmid = {40611420},
issn = {1875-5305},
abstract = {Bacteriophages, or phages, have emerged as powerful platforms in synthetic biology, offering innovative solutions for therapeutic and environmental challenges through advanced genome redesign strategies. This review explores a wide range of phage engineering techniques, including CRISPR (clustered regularly-interspaced short palindromic repeats)-Cas systems, phage display, random and site-directed mutagenesis, retrons, and rebooting approaches, highlighting their potential to create phages with tailored functionalities. CRISPR-Cas systems enable precise genome editing, allowing the development of phages with expanded host ranges, biofilm degradation capabilities, and targeted antimicrobial activity. Phage display facilitates the presentation of peptides on phage surfaces, enabling applications in targeted drug delivery, tumor imaging, and bioremediation. Beyond these, techniques like retron-mediated recombination and homologous recombination offer additional avenues for precise phage genome modification. In the therapeutic realm, engineered phages show promise in combating drug-resistant infections, modulating the microbiome, and delivering targeted therapies for cancer and other diseases. Environmentally, phage-based strategies, such as the use of phage-displayed metal-binding peptides, provide innovative solutions for bioremediation and reducing exposure to toxic heavy metals. This review also addresses challenges, such as phage resistance, immune responses, and the limitations of current engineering methods, while exploring future directions, including the development of improved CRISPR systems, phage-based biosensors, and high-throughput screening platforms. By integrating cutting-edge genome redesign strategies with diverse applications, this review underscores the transformative potential of engineered bacteriophages in addressing global healthcare and environmental sustainability challenges.},
}

@article {pmid40611351,
year = {2025},
author = {Smidler, AL and Akbari, OS},
title = {CRISPR technologies for the control and study of malaria-transmitting anopheline mosquitoes.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {252},
pmid = {40611351},
issn = {1756-3305},
support = {R01AI151004/GF/NIH HHS/United States ; R01AI151004/GF/NIH HHS/United States ; },
mesh = {*Anopheles/genetics/parasitology/physiology ; Animals ; *Mosquito Control/methods ; *Mosquito Vectors/genetics/parasitology/physiology ; *CRISPR-Cas Systems ; *Malaria/transmission/prevention & control ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Female ; Animals, Genetically Modified ; },
abstract = {Malaria is one of the deadliest diseases on the planet, killing approximately 600,000 people annually, and is transmitted by the bite of an anopheline mosquito. Anophelines, and the diseases they transmit, have changed the course of history and the fate of nations, and their successful control promises to end the transmission of malaria. With the advent of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technologies, the study and control of these deadly pests have been revolutionized. As the release of genetically modified anophelines is being considered, here we outline the advances in CRISPR/Cas9 technologies and how they have revolutionized the study of anopheline basic biology and the development of innovative vector control strategies. We outline the major findings of CRISPR-based basic biological research into traits relevant for vector control including, but not limited to, olfaction, chemosensation, neurobiology, and reproduction. Further, we summarize the advancements in CRISPR-based innovative vector control strategies, such as the precision-guided sterile insect technique (pgSIT), inherited female elimination by genetically encoded nucleases to interrupt alleles (IFEGENIA), X-shredder, Y-linked editors, and gene drives. All in all, this review summarizes the basic biological and vector control research undertaken using CRISPR since its advent approximately a decade ago.},
}

*Anopheles/genetics/parasitology/physiology
Animals
*Mosquito Control/methods
*Mosquito Vectors/genetics/parasitology/physiology
*CRISPR-Cas Systems
*Malaria/transmission/prevention & control
Gene Editing
Clustered Regularly Interspaced Short Palindromic Repeats
Humans
Female
Animals, Genetically Modified

@article {pmid40610423,
year = {2025},
author = {Fuhrmeister, ER and Kim, S and Mairal, SA and McCormack, C and Chieng, B and Swarthout, JM and Paulos, AH and Njenga, SM and Pickering, AJ},
title = {Context-Seq: CRISPR-Cas9 targeted nanopore sequencing for transmission dynamics of antimicrobial resistance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5898},
pmid = {40610423},
issn = {2041-1723},
support = {OPP1129535//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; UL1TR002544//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1906957//National Science Foundation (NSF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Nanopore Sequencing/methods ; *Drug Resistance, Bacterial/genetics ; Kenya ; Anti-Bacterial Agents/pharmacology ; Dogs ; *Bacteria/genetics/drug effects ; Klebsiella pneumoniae/genetics/drug effects ; Child ; Adult ; Escherichia coli/genetics/drug effects ; Poultry/microbiology ; beta-Lactamases/genetics ; Genome, Bacterial ; Haemophilus influenzae/genetics/drug effects ; },
abstract = {Precisely understanding how and to what extent antimicrobial resistance (AMR) is exchanged between animals and humans is needed to inform control strategies. Metagenomic sequencing has low detection for rare targets such as antibiotic resistance genes, while whole genome sequencing of isolates misses exchange between uncultured bacterial species. We introduce Context-Seq, CRISPR-Cas9 targeted sequencing of ARGs and their genomic context with long-reads. Using Context-Seq, we investigate genetically similar AMR elements containing the ARGs blaCTX-M and blaTEM between adults, children, poultry, and dogs in Nairobi, Kenya. We identify genetically distinct clusters containing blaTEM and blaCTX-M that are shared between animals and humans within and between households. We also uncover potentially pathogenic hosts of ARGs including Escherichia coli, Klebsiella pneumoniae, and Haemophilus influenzae in this study context. Context-Seq complements conventional methods to obtain an additional view of bacterial and mammalian hosts in the proliferation of AMR.},
}

*CRISPR-Cas Systems/genetics
Animals
Humans
*Nanopore Sequencing/methods
*Drug Resistance, Bacterial/genetics
Kenya
Anti-Bacterial Agents/pharmacology
Dogs
*Bacteria/genetics/drug effects
Klebsiella pneumoniae/genetics/drug effects
Child
Adult
Escherichia coli/genetics/drug effects
Poultry/microbiology
beta-Lactamases/genetics
Genome, Bacterial
Haemophilus influenzae/genetics/drug effects

@article {pmid40610421,
year = {2025},
author = {Sek, K and Chen, AXY and Cole, T and Armitage, JD and Tong, J and Yap, KM and Munoz, I and Dunbar, PA and Wu, S and van Elsas, MJ and Hidajat, O and Scheffler, C and Giuffrida, L and Henderson, MA and Meyran, D and Souza-Fonseca-Guimaraes, F and Nguyen, D and Huang, YK and de Menezes, MN and Derrick, EB and Chan, CW and Todd, KL and Chan, JD and Li, J and Lai, J and Petley, EV and Mardiana, S and Bosco, A and Waithman, J and Parish, IA and Mølck, C and Stewart, GD and Kats, L and House, IG and Darcy, PK and Beavis, PA},
title = {Tumor site-directed A1R expression enhances CAR T cell function and improves efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6123},
pmid = {40610421},
issn = {2041-1723},
support = {836379//Cancer Council Victoria/ ; },
mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment/immunology ; Cell Line, Tumor ; Female ; Receptor, Adenosine A2A/genetics/metabolism ; Mice, Inbred C57BL ; },
abstract = {The efficacy of Chimeric Antigen Receptor T cells against solid tumors is limited by immunosuppressive factors in the tumor microenvironment including adenosine, which suppresses Chimeric Antigen Receptor T cells through activation of the A2A receptor. To overcome this, Chimeric Antigen Receptor T cells are engineered to express A1 receptor, a receptor that signals inversely to A2A receptor. Using murine and human Chimeric Antigen Receptor T cells, constitutive A1 receptor overexpression significantly enhances Chimeric Antigen Receptor T cell effector function albeit at the expense of Chimeric Antigen Receptor T cell persistence. Through a CRISPR/Cas9 homology directed repair "knock-in" approach we demonstrate that Chimeric Antigen Receptor T cells engineered to express A1 receptor in a tumor-localized manner, enhances anti-tumor therapeutic efficacy. This is dependent on the transcription factor IRF8 and is transcriptionally unique when compared to A2A receptor deletion. This data provides a novel approach for enhancing Chimeric Antigen Receptor T cell efficacy in solid tumors and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation.},
}

Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/metabolism/immunology
*Neoplasms/therapy/immunology/genetics
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/metabolism
CRISPR-Cas Systems
Tumor Microenvironment/immunology
Cell Line, Tumor
Female
Receptor, Adenosine A2A/genetics/metabolism
Mice, Inbred C57BL

@article {pmid40610153,
year = {2025},
author = {Ren, W and Li, M and Liu, X and You, W and You, Q and Li, B and Ye, H and Zhang, R},
title = {Specific detection of DNA and RNA by the CRISPR-Cas12a system containing spacer split crRNA.},
journal = {Analytica chimica acta},
volume = {1367},
number = {},
pages = {344204},
doi = {10.1016/j.aca.2025.344204},
pmid = {40610153},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/analysis/genetics ; *RNA/analysis/genetics ; MicroRNAs/analysis/genetics ; *CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: The CRISPR/Cas12a system has emerged as a versatile molecular diagnostic tool due to its dual cis- and trans-cleavage activities. However, two key limitations hinder its broad application: high tolerance to single-base mismatches in DNA targets and strict reliance on DNA activators. To address these challenges, we hypothesized that structural reengineering of crRNA could enhance specificity and functional versatility. This study aimed to develop a modified Cas12a system capable of detecting DNA and RNA targets with improved single-base resolution, thereby expanding its utility in molecular diagnostics and clinical subclassification.

RESULTS: We engineered split crRNAs by introducing a split site within the spacer region, creating a spacer-split crRNA-activated Cas12a system (SPCas12a). This system exhibited three key advantages: First, SPCas12a demonstrated significantly enhanced specificity in discriminating single-base mutations compared to conventional full-sized crRNA systems. Second, it bypassed the DNA activator requirement, enabling direct detection of miRNA targets without reverse transcription. In addition, AlphaFold Server predictive structural modeling analysis showed that the split site selected by SPCas12a gives the Cas12a complex an open structural domain, which is conducive to the stable function of Cas12a. Third, integration with isothermal amplification enabled constructing an "AND" logic gate detection platform that processes multiple inputs within 40 min. As a proof-of-concept, SPCas12a successfully distinguished triple-negative breast cancer (TNBC) subtype cell lines by analyzing miRNA-210 and miRNA-21 biomarkers in different cell lines.

SIGNIFICANCE: SPCas12a overcomes fundamental limitations of current CRISPR diagnostics by unifying high-specificity DNA mutation detection and direct RNA sensing in a single platform. The split-crRNA design principle provides a universally adaptable strategy to enhance CRISPR-Cas systems, with immediate applications in precision oncology and infectious disease stratification where base-level discrimination and multi-target detection are critical.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA/analysis/genetics
*RNA/analysis/genetics
MicroRNAs/analysis/genetics
*CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40609997,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {804-817},
doi = {10.1134/S0006297925600139},
pmid = {40609997},
issn = {1608-3040},
mesh = {*RNA Interference ; *CRISPR-Cas Systems ; *Plant Diseases/virology/genetics/prevention & control ; Gene Editing/methods ; *Plants/virology/genetics ; *Disease Resistance/genetics ; *Biotechnology/methods ; Plant Viruses/genetics ; },
abstract = {Established genome editing technologies, such as CRISPR-Cas and RNA interference (RNAi), have significantly advanced research studies in nearly all fields of life sciences, including biotechnology and medicine, and have become increasingly in demand in plant biology. In the review, we present the main principles of the CRISPR-Cas and RNAi technologies and their application in model plants and crops for the control of viral diseases. The review explores the antiviral effects they provide, including direct suppression of genomes of DNA- and RNA-containing viruses and inhibition of activity of host genes that increase plant susceptibility to viruses. We also provide a detailed comparison of the effectiveness of CRISPR-Cas and RNAi methods in plant protection, as well as discuss their advantages and disadvantages, factors limiting their application in practice, and possible approaches to overcome such limitations.},
}

*RNA Interference
*CRISPR-Cas Systems
*Plant Diseases/virology/genetics/prevention & control
Gene Editing/methods
*Plants/virology/genetics
*Disease Resistance/genetics
*Biotechnology/methods
Plant Viruses/genetics

@article {pmid40609996,
year = {2025},
author = {Kazakova, AA and Leonova, EI and Sopova, JV and Chirinskaite, AV and Minskaya, ES and Kukushkin, IS and Ivanov, RA and Reshetnikov, VV},
title = {Progress in CRISPR/CAS13-Mediated Suppression of Influenza A and SARS-CoV-2 Virus Infection in in vitro and in vivo Models.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {786-803},
doi = {10.1134/S0006297925601212},
pmid = {40609996},
issn = {1608-3040},
mesh = {*CRISPR-Cas Systems ; Animals ; *SARS-CoV-2/genetics ; Humans ; *COVID-19/therapy/virology ; *Influenza A virus/genetics ; *Influenza, Human/therapy/virology/genetics ; Disease Models, Animal ; RNA, Viral/genetics/metabolism ; Antiviral Agents ; },
abstract = {The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental in vitro and in vivo studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.},
}

*CRISPR-Cas Systems
Animals
*SARS-CoV-2/genetics
Humans
*COVID-19/therapy/virology
*Influenza A virus/genetics
*Influenza, Human/therapy/virology/genetics
Disease Models, Animal
RNA, Viral/genetics/metabolism
Antiviral Agents

@article {pmid40609995,
year = {2025},
author = {Volodina, OV and Demchenko, AG and Anuchina, AA and Ryzhkova, OP and Kovalskaya, VA and Kondrateva, EV and Tabakov, VY and Lavrov, AV and Smirnikhina, SA},
title = {Selection of Optimal pegRNAs to Enhance Efficiency of Prime Editing in AT-Rich Genome Regions.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {6},
pages = {773-785},
doi = {10.1134/S0006297924604672},
pmid = {40609995},
issn = {1608-3040},
mesh = {*Gene Editing/methods ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems ; },
abstract = {Prime editing is a highly promising strategy for treating hereditary disorders due to its superior efficiency and safety profile compared to the conventional CRISPR-Cas9 systems. This study is dedicated to development of a causal therapy for cystic fibrosis by targeting the F508del variant of the CFTR gene using prime editing, as this specific deletion accounts for a substantial proportion of cystic fibrosis cases. While prime editing has shown remarkable precision in introducing targeted genetic modifications, its application in AT-rich genomic regions, such as the one containing the F508del variant, remains challenging. To overcome this limitation, we systematically evaluated 24 pegRNAs designed for two distinct prime editing systems, PEmax and PE2-NG. Efficiency of the F508del variant correction reached 2.81% (without normalization for transfection efficiency) in the airway basal cells from the patients with homozygous F508del mutation. However, the average transfection efficiency was only 11.9%, emphasizing critical need for the advancements in delivery methodologies. These findings highlight potential of prime editing as an approach for treating cystic fibrosis, while also underscoring necessity for further optimization of both editing constructs and delivery vectors to achieve clinically relevant correction levels.},
}

*Gene Editing/methods
Humans
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Cystic Fibrosis/genetics/therapy
CRISPR-Cas Systems

@article {pmid40609268,
year = {2025},
author = {Allan, AC and Scott, B and Tate, W and Jameson, PE and MacRae, E and Conner, AJ and Drummond, R and Foster, A and Martin, C and Caradus, J},
title = {Human-mediated outdoor genome editing is not possible so therefore poses no risk to the environment.},
journal = {Ecotoxicology and environmental safety},
volume = {302},
number = {},
pages = {118609},
doi = {10.1016/j.ecoenv.2025.118609},
pmid = {40609268},
issn = {1090-2414},
abstract = {There is a world-wide re-examination of the regulations that surround genetic technologies, including gene edited organisms. In many countries, crop plants with small gene edits and where no foreign DNA is introduced are exempt from detailed regulatory assessment. This will allow these types of plants to be released, after assessment for benefit and risk, by plant breeders or plant scientists. The full regulatory risk assessment and risk management of novel (including transgenic) plants is well established and focusses on five key criteria (weediness, gene flow, plant pests, non-target impact, biodiversity). However, plants produced by "traditional" plant breeding technologies, many of which have been subject to random mutagenesis or wide crosses that may introduce enormous numbers of DNA changes, are almost never considered novel so are not assessed for risk through a regulatory system. The most targeted, versatile and widely used gene editing technique involves the enzyme-RNA complex, CRISPR-Cas. This method can produce far more precise and targeted changes than "traditional" mutagenesis techniques. Getting the CRISPR-Cas machinery into plant or animal cells requires highly sterile tissue culture and sophisticated delivery tools. Therefore, gene editing, in the open environment by "field spraying", is not currently possible. While other uses of nucleotide chemistry - such as double stranded RNA - have been applied to plants to knock down gene expression, this is not gene editing and produces no DNA change. Suggestions that gene editing using CRISPR-Cas can occur through spraying directly on to plants in the outside environment is fanciful, incorrect and misleading.},
}

@article {pmid40608406,
year = {2025},
author = {Ito, K and Ito, Y},
title = {Comparative genomic analysis of Latilactobacillus sakei strains provides new insights into their association with different niche adaptations.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {7},
pages = {},
doi = {10.1099/mic.0.001578},
pmid = {40608406},
issn = {1465-2080},
mesh = {*Genome, Bacterial ; Phylogeny ; *Adaptation, Physiological/genetics ; Plasmids/genetics ; *Latilactobacillus sakei/genetics/classification/physiology/growth & development/metabolism ; Genomics ; Carbohydrate Metabolism/genetics ; Fermentation ; Genetic Variation ; },
abstract = {Latilactobacillus sakei, a lactic acid bacterium in diverse environments such as fermented foods, meat and the human gastrointestinal tract, exhibits significant genetic diversity and niche-specific adaptations. This study conducts a comprehensive comparative genomic analysis of 29 complete L. sakei genomes to uncover the genetic mechanisms underlying these adaptations. Phylogenetic analysis divided the species into three distinct clades that did not correlate with the source of isolation and did not suggest any niche-specific evolutionary direction. The pan-genome analysis revealed a substantial core genome alongside a diverse genetic repertoire, indicating both high genetic conservation and adaptability. Predicted growth rates based on codon use bias analysis suggest that L. sakei strains have an overall faster growth rate and may be able to efficiently dominate in competitive environments. Plasmid analysis revealed a variety of plasmids carrying genes essential for carbohydrate metabolism, enhancing L. sakei's ability to thrive in various fermentation substrates. It was also found that the number of genes belonging to the GH1 family amongst sugar metabolism-related genes present on chromosomes and plasmids varies between strains and that AA1, which is involved in alcohol oxidation, has been acquired from plasmids. blast analysis revealed that some strains have environmental adaptation gene clusters of cell surface polysaccharides that may mediate attachment to food and mucosa. The knowledge gleaned from this study lays a solid foundation for future research aimed at harnessing the genetic traits of L. sakei strains for industrial and health-related applications.},
}

*Genome, Bacterial
Phylogeny
*Adaptation, Physiological/genetics
Plasmids/genetics
*Latilactobacillus sakei/genetics/classification/physiology/growth & development/metabolism
Genomics
Carbohydrate Metabolism/genetics
Fermentation
Genetic Variation

@article {pmid40608358,
year = {2025},
author = {Li, YG and Haeusser, D and Margolin, W and Christie, PJ},
title = {Conjugative delivery of toxin genes ccdB and kil confers synergistic killing of bacterial recipients.},
journal = {Journal of bacteriology},
volume = {},
number = {},
pages = {e0016825},
doi = {10.1128/jb.00168-25},
pmid = {40608358},
issn = {1098-5530},
abstract = {The bacterial type IV secretion systems (T4SS) are medically problematic for their roles in the dissemination of mobile genetic elements or effector proteins, but they also have great potential for new antimicrobial therapies. Recent studies have deployed the T4SS subfamily of conjugation systems to deliver gene editing CRISPR/Cas systems to disrupt drug resistance genes or kill targeted bacterial recipients. However, the therapeutic potential of conjugative CRISPR/Cas delivery is compromised by mutations or host repair systems that diminish the efficiency with which CRISPR/Cas induces double-strand breaks in new transconjugants. Here, we compared the efficiencies of conjugation-based killing systems based on the delivery of CRISPR-Cas9 elements or toxin genes encoding the bacteriophage lambda Kil peptide or the F plasmid-encoded CcdB. Escherichia coli equipped with one of two efficient conjugation systems, pKM101 (IncN) or F (IncF), served as donors to mobilize plasmids carrying the cognate oriT sequence and one or more toxic elements. Overall, toxin gene delivery proved significantly more effective than CRISPR-Cas9 in killing of transconjugant population, but the highest levels of growth suppression of both E. coli and Klebsiella pneumoniae recipients were achieved by a combination of CRISPR-Cas9 plus one or two toxin genes. By contrast, capsule production conferred no or very slight protective effects on plasmid acquisition and killing of either species. We propose that the conjugative co-transfer of two or more toxic elements with distinct mechanisms of action has strong potential for growth suppression of targeted species in environmental or clinical settings.IMPORTANCEThe prevalence of antibiotic resistance emphasizes the need for alternative antimicrobial intervention strategies. We engineered Escherichia coli for conjugative transmission of plasmids encoding CRISPR-Cas9 elements or genes encoding the cell division inhibitor Kil or gyrase poisoner CcdB. Delivery of toxin genes more effectively suppressed the growth of E. coli recipients than CRISPR-Cas9, but the combinatorial delivery of CRISPR-Cas9 and a toxin gene or two toxin genes elicited the strongest killing effects. Capsule production by E. coli or Klebsiella pneumoniae recipient cells had no or little protective effect on plasmid acquisition or growth suppression. Our findings suggest that probiotic donor strains equipped for conjugative delivery of two or more toxic elements may prove effective as an alternative or adjunct to traditional antimicrobials.},
}

@article {pmid40607431,
year = {2025},
author = {Wang, JW and Liu, JH and Xun, JJ},
title = {CCR5 gene editing and HIV immunotherapy: current understandings, challenges, and future directions.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1590690},
pmid = {40607431},
issn = {1664-3224},
mesh = {*Receptors, CCR5/genetics/immunology ; Humans ; *Gene Editing/methods ; *HIV Infections/therapy/immunology/genetics ; *Immunotherapy/methods ; *HIV-1/immunology ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Human immunodeficiency virus (HIV) infection remains a major global public health challenge. Although highly active antiretroviral therapy (HAART or ART) can effectively control viral replication, it fails to eradicate latent viral reservoirs and poses limitations such as lifelong medication and cumulative drug toxicity. This study focuses on the pivotal role of C-C chemokine receptor 5 (CCR5) gene editing in HIV immunotherapy, particularly highlighting the natural resistance to R5-tropic HIV strains observed in the "Berlin" and "London" patients carrying the homozygous CCR5-Δ32 mutation. We further explore the synergistic potential of multiplex gene editing strategies-including CCR5, CXCR4, and HIV LTR loci-and the combinatorial mechanisms between gene editing technologies and immunotherapy. A personalized treatment framework is proposed to address the clinical heterogeneity among people living with HIV. In addition, we assess the balance between long-term safety and global accessibility of gene-editing approaches such as CRISPR/Cas9, emphasizing strategies to enhance therapeutic efficacy while reducing cost and off-target effects. Our findings suggest that the integration of CCR5-targeted gene editing with immune-based interventions holds great promise for overcoming current therapeutic limitations and achieving functional HIV cure. However, key challenges-such as immune rejection, viral tropism switching, and economic feasibility-must be resolved. This integrative approach provides a robust theoretical and technical foundation for the next generation of HIV treatment paradigms.},
}

*Receptors, CCR5/genetics/immunology
Humans
*Gene Editing/methods
*HIV Infections/therapy/immunology/genetics
*Immunotherapy/methods
*HIV-1/immunology
CRISPR-Cas Systems
Genetic Therapy/methods

@article {pmid40605043,
year = {2025},
author = {Salazar-García, LM and Damas-Ramos, LC and Trejo-Alarcón, LM and Rago, D and Ahonen, L and Cruz-Morales, P and Ponce-Noyola, P and Licona-Cassani, C},
title = {CRISPR-driven enhanced hydrocarbon emulsification in an environmental Pseudomonas aeruginosa strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {151},
pmid = {40605043},
issn = {1475-2859},
support = {NNF20CC0035580//Novo Nordisk Fonden/ ; },
mesh = {*Pseudomonas aeruginosa/metabolism/genetics/isolation & purification ; Glycolipids/metabolism/biosynthesis ; *CRISPR-Cas Systems ; *Hydrocarbons/metabolism/chemistry ; Bacterial Proteins/genetics/metabolism ; Pyocyanine/metabolism/biosynthesis ; Emulsions ; Gene Editing ; Sigma Factor/genetics ; Petroleum Pollution ; },
abstract = {BACKGROUND: Oil spills are a major concern due to the economic impact and severe effects on the ecosystem. To mitigate oil spills, hydrocarbon dispersion through emulsification is a promising approach, as it makes oil more susceptible to degradation by microorganisms. Environmental strains of Pseudomonas aeruginosa have demonstrated significant potential for producing rhamnolipids (RMLs) and pyocyanin (PYO), secondary metabolites associated to hydrocarbon emulsification. In this study, we isolated and characterized an environmental strain from an oil-contaminated site in the Gulf of Mexico. Upon genome sequencing and taxonomic classification, we developed genetic engineering tools and assessed their capacity to produce PYO and RMLs, molecules relevant for hydrocarbon emulsification.

RESULTS: Using the CRISPR/Cas9-APOBEC1-UGI system, we generated a targeted cytosine to thymine transition in the rpoS gene to generate a premature STOP codon. The resulting mutant exhibited increased production of PYO and RMLs, along with enhanced gasoline emulsification in cell-free supernatants, demonstrating successful modulation of a key regulatory gene. While the strain IGLPR01 retains certain virulence-associated features, this study contributes to the exploration of environmental isolates as future candidate chassis for biosurfactant production, emphasizing the need for further safety evaluation and rational attenuation strategies.

CONCLUSION: This study provides a successful example of implementing CRISPR-based editing in an environmental P. aeruginosa strain. Despite the technical challenges, a genetic editing system was established and validated through a proof of concept to increase production of relevant metabolites. Our work demonstrates the applicability of genetic engineering tools in non-model environmental isolates, facilitating further developments. Importantly, the presence of virulence-associated features highlights the need for in-depth evaluation of pathogenicity and containment strategies before considering any future biotechnological applications.},
}

*Pseudomonas aeruginosa/metabolism/genetics/isolation & purification
Glycolipids/metabolism/biosynthesis
*CRISPR-Cas Systems
*Hydrocarbons/metabolism/chemistry
Bacterial Proteins/genetics/metabolism
Pyocyanine/metabolism/biosynthesis
Emulsions
Gene Editing
Sigma Factor/genetics
Petroleum Pollution

@article {pmid40605002,
year = {2025},
author = {Chou, J and Esmaeili Anvar, N and Elghaish, R and Chen, J and Hart, T},
title = {Z-scores outperform similar methods for analyzing CRISPR paralog synthetic lethality screens.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {188},
pmid = {40605002},
issn = {1474-760X},
support = {R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; R35GM130119/GM/NIGMS NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; R35CA274234/CA/NCI NIH HHS/United States ; P30CA16672/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Synthetic Lethal Mutations ; Cell Line, Tumor ; },
abstract = {Genetic screens offer a promising strategy for identifying tumor-specific therapeutic targets, but single-gene knockout screens often miss functionally redundant paralogs. Multiplex Cas9 and Cas12a CRISPR systems have been deployed to assay genetic interactions, but analysis pipelines vary considerably. Here we evaluate data from four in4mer CRISPR/Cas12a screens in cancer cell lines, using delta log fold change, Z-transformed dLFC, and rescaled dLFC approaches to identify synthetic lethal interactions. Both ZdLFC and RdLFC provide more consistent identification of synthetic lethal pairs across cell lines compared to the unscaled dLFC method, while ZdLFC benefits from not requiring a training set of known interactors.},
}

Humans
*CRISPR-Cas Systems
*Synthetic Lethal Mutations
Cell Line, Tumor

@article {pmid40577485,
year = {2025},
author = {Rissone, A and La Spina, M and Bresciani, E and Syed, ZA and Combs, CA and Kirby, M and Elkahloun, A and Chen, V and Sood, R and Burgess, SM and Puertollano, R},
title = {The transcription factors Tfeb and Tfe3 are required for survival and embryonic development of pancreas and liver in zebrafish.},
journal = {PLoS genetics},
volume = {21},
number = {6},
pages = {e1011754},
pmid = {40577485},
issn = {1553-7404},
mesh = {Animals ; *Zebrafish/genetics/embryology/growth & development ; *Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Pancreas/embryology/metabolism/growth & development ; *Liver/embryology/metabolism/growth & development ; *Embryonic Development/genetics ; Autophagy/genetics ; Gene Expression Regulation, Developmental ; Apoptosis/genetics ; Oxidative Stress/genetics ; CRISPR-Cas Systems ; },
abstract = {The transcription factors TFEB and TFE3 modulate expression of lysosomal, autophagic, and metabolic genes to restore energy and cellular homeostasis in response to a variety of stress conditions. Since their role during vertebrate development is less characterized, we used CRISPR/Cas9 to deplete tfeb, tfe3a, and tfe3b in zebrafish. The simultaneous lack of these genes compromised embryo survival during early development, with an almost complete lethality of the larvae by 8-10 dpf. The knockout animals showed apoptosis in brain and retina and alterations in pancreas, liver, and gut. Exocrine pancreas presented the most severe defects, with accumulation of abnormal zymogen granules leading to acinar atrophy in embryos and pancreatitis-like phenotypes in adults; likely due to a block of the autophagy machinery implicated in removal of damaged granules. Knockout animals displayed increased susceptibility to oxidative and heat-shock stress. Our work reveals an essential role of Tfeb and Tfe3 in maintaining cellular and tissue homeostasis during development.},
}

Animals
*Zebrafish/genetics/embryology/growth & development
*Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
*Pancreas/embryology/metabolism/growth & development
*Liver/embryology/metabolism/growth & development
*Embryonic Development/genetics
Autophagy/genetics
Gene Expression Regulation, Developmental
Apoptosis/genetics
Oxidative Stress/genetics
CRISPR-Cas Systems

@article {pmid40516528,
year = {2025},
author = {Matthews, RE and Danac, JMC and Naden, EL and Farleigh Smith, LE and Lestari, S and Gungi, A and Appert, A and Buttress, T and Verma, A and Sinclair, O and Chong, F and Suberu, J and Antrobus, R and Bonev, B and Dawson, MA and Reid, AJ and Timms, RT and Ahringer, J and Tchasovnikarova, IA},
title = {CRAMP1 drives linker histone expression to enable Polycomb repression.},
journal = {Molecular cell},
volume = {85},
number = {13},
pages = {2503-2516.e8},
doi = {10.1016/j.molcel.2025.05.031},
pmid = {40516528},
issn = {1097-4164},
mesh = {*Histones/genetics/metabolism ; *Polycomb Repressive Complex 2/genetics/metabolism ; Animals ; Humans ; Mice ; Promoter Regions, Genetic ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Epigenetic Repression ; },
abstract = {In contrast to the well-understood role of core histones in DNA packaging, the function of the linker histone (H1) remains enigmatic. Challenging the prevailing view that linker histones are a general feature of heterochromatin, here we show a critical requirement for H1 in Polycomb repressive complex 2 (PRC2) function. A CRISPR-Cas9 genetic screen using a fluorescent PRC2 reporter identified an essential role for the poorly characterized gene CRAMP1 in PRC2-mediated repression. CRAMP1 localizes to the promoters of expressed H1 genes and positively regulates their transcription. CRAMP1 ablation simultaneously depletes all linker histones, which results in selective decompaction of H3K27me3-marked loci and derepression of PRC2 target genes without concomitant loss of PRC2 occupancy or enzymatic activity. Strikingly, we find that linker histones preferentially localize to genomic loci marked by H3K27me3 across diverse cell types and organisms. Altogether, these data demonstrate a prominent role for linker histones in epigenetic repression by PRC2.},
}

*Histones/genetics/metabolism
*Polycomb Repressive Complex 2/genetics/metabolism
Animals
Humans
Mice
Promoter Regions, Genetic
Heterochromatin/genetics/metabolism
CRISPR-Cas Systems
*Epigenetic Repression

@article {pmid40399675,
year = {2025},
author = {Han, M and Fu, ML and Zhu, Y and Choi, AA and Li, E and Bezney, J and Cai, S and Miles, L and Ma, Y and Qi, LS},
title = {Programmable control of spatial transcriptome in live cells and neurons.},
journal = {Nature},
volume = {643},
number = {8070},
pages = {241-251},
pmid = {40399675},
issn = {1476-4687},
mesh = {Animals ; *Neurons/metabolism/cytology ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; Mice ; Neurites/metabolism ; Humans ; RNA Transport/genetics ; Ribosomes/metabolism ; Neuronal Outgrowth/genetics ; Actins/genetics/metabolism ; Rats ; Protein Biosynthesis ; Cells, Cultured ; Microtubules/metabolism ; Female ; Axons/metabolism ; },
abstract = {Spatial RNA organization has a pivotal role in diverse cellular processes and diseases[1-4]. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions[1,5]. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells. CRISPR-TO enables targeted localization of endogenous RNAs to diverse subcellular compartments, including the outer mitochondrial membrane, p-bodies, stress granules, telomeres and nuclear stress bodies, across various cell types. It allows for inducible and reversible bidirectional RNA transport along microtubules via motor proteins, facilitating real-time manipulation and monitoring of RNA localization dynamics in living cells. In primary cortical neurons, we demonstrate that repositioned mRNAs undergo local translation along neurites and at neurite tips, and co-transport with ribosomes, with β-actin mRNA localization enhancing the formation of dynamic filopodial protrusions and inhibiting axonal regeneration. CRISPR-TO-enabled screening in primary neurons identifies Stmn2 mRNA localization as a driver of neurite outgrowth. By enabling large-scale perturbation of the spatial transcriptome, CRISPR-TO bridges a critical gap left by sequencing and imaging technologies, offering a versatile platform for high-throughput functional interrogation of RNA localization in living cells and organisms.},
}

Animals
*Neurons/metabolism/cytology
*Transcriptome/genetics
*CRISPR-Cas Systems/genetics
RNA, Messenger/metabolism/genetics
Mice
Neurites/metabolism
Humans
RNA Transport/genetics
Ribosomes/metabolism
Neuronal Outgrowth/genetics
Actins/genetics/metabolism
Rats
Protein Biosynthesis
Cells, Cultured
Microtubules/metabolism
Female
Axons/metabolism

@article {pmid40359943,
year = {2025},
author = {Wang, Y and Wu, J and Yang, S and Li, X and Wang, J and Lv, Q and Zhu, X and Lu, G and Zhang, J and Shen, WH and Liu, B and Lin, J and Dong, A},
title = {Structural and functional interrelationships of histone H2A with its variants H2A.Z and H2A.W in Arabidopsis.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {7},
pages = {1240-1249.e5},
doi = {10.1016/j.str.2025.04.015},
pmid = {40359943},
issn = {1878-4186},
mesh = {*Histones/chemistry/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/chemistry/metabolism/genetics ; Nucleosomes/metabolism/chemistry ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; Mutation ; Protein Isoforms/chemistry/metabolism/genetics ; },
abstract = {Multiple histone H2A variants are known in eukaryotes. However, the functional relationship between H2A and its variants in plants remains largely obscure. Using CRISPR-Cas9 editing, we generated a mutant lacking four H2A isoforms in Arabidopsis and analyzed the functional and structural relationships between H2A, H2A.Z, and H2A.W. RNA sequencing and phenotype analyses revealed mild changes in gene transcription and plant development in mutants lacking H2A, H2A.Z, or H2A.W compared with the wild-type plants. Chromatin immunoprecipitation sequencing analysis showed that H2A can substitute for both H2A.Z and H2A.W across the genome, including in euchromatin and heterochromatin regions. However, H2A.Z replaced both H2A and H2A.W primarily within the euchromatin regions. By using DNA and histones from Arabidopsis, we constructed nucleosomes containing H2A, H2A.Z, or H2A.W and resolved their cryogenic electron microscopy (cryo-EM) structures at near-atomic resolution. Collectively, the results reveal the structural similarity and functional redundancy of H2A and its variants in Arabidopsis.},
}

*Histones/chemistry/genetics/metabolism
*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/chemistry/metabolism/genetics
Nucleosomes/metabolism/chemistry
Cryoelectron Microscopy
CRISPR-Cas Systems
Mutation
Protein Isoforms/chemistry/metabolism/genetics

@article {pmid40344384,
year = {2025},
author = {Yao, Z and Li, W and He, K and Wang, H and Xu, Y and Wu, Q and Wang, L and Nie, Y},
title = {Facilitating crRNA Design by Integrating DNA Interaction Features of CRISPR-Cas12a System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {25},
pages = {e2501269},
pmid = {40344384},
issn = {2198-3844},
support = {32172175//National Natural Science Foundation of China/ ; 32172307//National Natural Science Foundation of China/ ; 2023C02006//Key R&D Program of Zhejiang/ ; KYCX24_2592//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; NO.111-2-06//Priority Academic Program Development of Jiangsu Higher Education Institutions, the 111 Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics/metabolism ; Molecular Dynamics Simulation ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR-Cas12a system has gained significant attention as a rapid nucleic acid diagnostic tool due to its crRNA-guided trans-cleavage activity. Accurately predicting the activity of different targets is significant to facilitate the crRNA availability but remains challenging. In this study, a novel approach is presented that combines molecular dynamics simulations and neural network modeling to predict the trans-cleavage activity. Unlike conventional tools that rely solely on the base sequences, our method integrated sequence features and molecular interaction features of DNA in the CRISPR-Cas12a system, significantly improving prediction accuracy. Through feature importance analysis, key sequence features that influence Cas12a trans-cleavage activity are identified. Additionally, a crRNA-DNA library with over 23 456 feature sequences from representative viruses and bacteria is established, and validated the high predictive accuracy of the model (Pearson's r = 0.9328) by screening crRNAs from reference targets. This study offers new insights into the molecular interactions of Cas12a/crRNA-DNA and provides a reliable framework for optimizing crRNA design, facilitating the application of the CRISPR-Cas12a in rapid nucleic acid diagnostics.},
}

*CRISPR-Cas Systems/genetics
*DNA/genetics/metabolism
Molecular Dynamics Simulation
*CRISPR-Associated Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40302201,
year = {2025},
author = {Hao, JH and Kang, X and Zhang, L and Chen, J and Wang, D and Dong, S and Li, X and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-Mediated SiEPF2 Mutagenesis Attenuates Drought Tolerance and Yield in Foxtail Millet (Setaria italica).},
journal = {Plant, cell & environment},
volume = {48},
number = {8},
pages = {6043-6046},
doi = {10.1111/pce.15597},
pmid = {40302201},
issn = {1365-3040},
support = {//This study was supported by the National Key Research and Development Programme of China (2023YFD1202702), National Natural Science Foundation (32400217 and 32200222) and Start-up Fund of Shanxi Agricultural University (2021BQ84)./ ; },
mesh = {*Setaria Plant/genetics/physiology/growth & development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Mutagenesis ; Plant Stomata/physiology ; Gene Expression Regulation, Plant ; Drought Resistance ; },
abstract = {Plants employ peptide ligands to coordinate development and integrate environmental signals via dedicated cascades (#ref-0013). Epidermal patterning factor (EPF), plays a significant role in regulating stomatal density, seed germination and panicle development (#ref-0008). EPF/EPFL enhance drought tolerance by reducing stomatal density have been reported in multiple species, including Hordeum vulgare, Arabidopsis thaliana, Populus spp., Vitis vinifera, Sorghum bicolor and Brassica napus (#ref-0001). Our previous study has shown that EPF can mediate drought resistance in foxtail millet by regulating stomatal density (#ref-0004). Furthermore, evidence suggests that plants can regulate photosynthesis through stomatal modification, ultimately enhancing yield (#ref-0005). Beyond stomatal density regulation, the EPF/EPFL gene family modulates seed germination through phytohormone signalling and regulates inflorescence development via ligand-receptor interactions (#ref-0007). OsEPFLs act as upstream ligands for the OsER1 receptor, activating the MAPK signalling cascade to regulate panicle morphogenesis (#ref-0002). In this study, we sought to elucidate how SiEPF2 balances drought resistance and yield in foxtail millet by modulating stomatal density and panicle morphology. Our findings not only provide novel insights into SiEPF2's role in abiotic stress responses but also contribute valuable genetic resources for high-yield breeding programmes in millet crops.},
}

*Setaria Plant/genetics/physiology/growth & development
Droughts
*Plant Proteins/genetics/metabolism/physiology
*CRISPR-Cas Systems/genetics
Mutagenesis
Plant Stomata/physiology
Gene Expression Regulation, Plant
Drought Resistance

@article {pmid40280813,
year = {2025},
author = {Hu, N and Tian, H and Li, Y and Li, X and Li, D and Li, L and Wang, S and Zhang, Y and Shi, X and Huang, B and Lu, Q and Wang, T and Pan, X and Tu, L and Dai, D and Zhang, B and Peng, R and Yan, F},
title = {pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1788-1808},
doi = {10.1016/j.tibtech.2025.03.016},
pmid = {40280813},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Solanum lycopersicum/genetics ; Arabidopsis/genetics ; DNA, Bacterial/genetics ; Promoter Regions, Genetic ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genome, Plant/genetics
Nicotiana/genetics
Plants, Genetically Modified/genetics
Solanum lycopersicum/genetics
Arabidopsis/genetics
DNA, Bacterial/genetics
Promoter Regions, Genetic

@article {pmid40263591,
year = {2025},
author = {Rohde, T and Demirtas, TY and Süsser, S and Shaw, AH and Kaulich, M and Billmann, M},
title = {BaCoN (Balanced Correlation Network) improves prediction of gene buffering.},
journal = {Molecular systems biology},
volume = {21},
number = {7},
pages = {807-824},
pmid = {40263591},
issn = {1744-4292},
support = {397484323 - TRR259//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Gene Regulatory Networks ; Cell Line, Tumor ; Algorithms ; Transcriptome ; *Computational Biology/methods ; Neoplasms/genetics ; },
abstract = {Buffering between genes, where one gene can compensate for the loss of another gene, is fundamental for robust cellular functions. While experimentally testing all possible gene pairs is infeasible, gene buffering can be predicted genome-wide under the assumption that a gene's buffering capacity depends on its expression level and its absence primes a severe fitness phenotype of the buffered gene. We developed BaCoN (Balanced Correlation Network), a post hoc unsupervised correction method that amplifies specific signals in expression-vs-fitness correlation networks. We quantified 147 million potential buffering relationships by associating CRISPR-Cas9-screening fitness effects with transcriptomic data across 1019 Cancer Dependency Map (DepMap) cell lines. BaCoN outperformed state-of-the-art methods, including multiple linear regression based on our compiled gene buffering prediction metrics. Combining BaCoN with batch correction or Cholesky data whitening further boosts predictive performance. We characterized 808 high-confidence buffering predictions and found that in contrast to buffering gene pairs overall, buffering paralogs were on different chromosomes. BaCoN performance increases with more screens and genes considered, making it a valuable tool for gene buffering predictions from the growing DepMap.},
}

Humans
CRISPR-Cas Systems
*Gene Regulatory Networks
Cell Line, Tumor
Algorithms
Transcriptome
*Computational Biology/methods
Neoplasms/genetics

@article {pmid40205048,
year = {2025},
author = {Fell, CW and Villiger, L and Lim, J and Hiraizumi, M and Tagliaferri, D and Yarnall, MTN and Lee, A and Jiang, K and Kayabolen, A and Krajeski, RN and Schmitt-Ulms, C and Ramani, H and Yousef, SM and Roberts, N and Vakulskas, CA and Nishimasu, H and Abudayyeh, OO and Gootenberg, JS},
title = {Reprogramming site-specific retrotransposon activity to new DNA sites.},
journal = {Nature},
volume = {642},
number = {8069},
pages = {1080-1089},
pmid = {40205048},
issn = {1476-4687},
mesh = {*Retroelements/genetics ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Finches/genetics ; *Gene Editing/methods ; *DNA/genetics ; Genome/genetics ; *Mutagenesis, Insertional/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Reverse Transcription ; },
abstract = {Retroelements have a critical role in shaping eukaryotic genomes. For instance, site-specific non-long terminal repeat retrotransposons have spread widely through preferential integration into repetitive genomic sequences, such as microsatellite regions and ribosomal DNA genes[1-6]. Despite the widespread occurrence of these systems, their targeting constraints remain unclear. Here we use a computational pipeline to discover multiple new site-specific retrotransposon families, profile members both biochemically and in mammalian cells, find previously undescribed insertion preferences and chart potential evolutionary paths for retrotransposon retargeting. We identify R2Tg, an R2 retrotransposon from the zebra finch, Taeniopygia guttata, as an orthologue that can be retargeted by payload engineering for target cleavage, reverse transcription and scarless insertion of heterologous payloads at new genomic sites. We enhance this activity by fusing R2Tg to CRISPR-Cas9 nickases for efficient insertion at new genomic sites. Through further screening of R2 orthologues, we select an orthologue, R2Tocc, with natural reprogrammability and minimal insertion at its natural 28S site, to engineer SpCas9[H840A]-R2Tocc, a system we name site-specific target-primed insertion through targeted CRISPR homing of retroelements (STITCHR). STITCHR enables the scarless, efficient installation of edits, ranging from a single base to 12.7 kilobases, gene replacement and use of in vitro transcribed or synthetic RNA templates. Inspired by the prevalence of nLTR retrotransposons across eukaryotic genomes, we anticipate that STITCHR will serve as a platform for scarless programmable integration in dividing and non-dividing cells, with both research and therapeutic applications.},
}

*Retroelements/genetics
Animals
Humans
CRISPR-Cas Systems/genetics
Finches/genetics
*Gene Editing/methods
*DNA/genetics
Genome/genetics
*Mutagenesis, Insertional/genetics
CRISPR-Associated Protein 9/metabolism/genetics
Mice
Reverse Transcription

@article {pmid40199626,
year = {2025},
author = {Lu, Y and Wang, J and Xu, Y and Xu, M and Li, B and Fan, Z and Liu, J and Li, X and Cai, Z and Zheng, Y and Wang, W and Yang, J and Zhang, Z and Liu, Z},
title = {Long-offset paired nicking-based efficient and precise strategy for in vivo targeted insertion.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1743-1764},
doi = {10.1016/j.tibtech.2025.02.020},
pmid = {40199626},
issn = {1879-3096},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; Genetic Therapy/methods ; Gene Knock-In Techniques/methods ; *Gene Targeting/methods ; DNA Repair ; Hepatocytes/metabolism ; Deoxyribonuclease I/metabolism/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-based targeted insertion of DNA fragments holds great promise for gene therapy. However, designing highly efficient and precise integration of large DNA segments in somatic cells while avoiding unpredictable products remains challenging. Here, we devised a novel long-offset paired nicking target integration (LOTI) strategy, which enhances the capacity of Cas9 nickase (Cas9n) in targeted gene integration in somatic cells, yielding higher knock-in (KI) efficiency compared with classical nickase-based approaches. The underlying repair mechanism involves the DNA repair proteins Rad51 and Rad52, and Ligase I/III. Moreover, we achieved efficient KI of at least 1.5-kb gene fragments in hepatocytes and recovery 55% FIX activity in a hemophilia B mouse model using only one-dose plasmid DNA delivery. Compared with the Cas9-based strategy, LOTI reduces off-target activity and restricts the formulation of unwanted insertions and deletions (indels) at the target site. Thus, LOTI provides a precise and efficient strategy for gene integration in somatic cells in vivo.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
Genetic Therapy/methods
Gene Knock-In Techniques/methods
*Gene Targeting/methods
DNA Repair
Hepatocytes/metabolism
Deoxyribonuclease I/metabolism/genetics

@article {pmid40192038,
year = {2025},
author = {Shi, K and Huang, W and Zhu, M and Teng, S and Zhang, J and Duan, Z and Zhu, C and Hu, T and Wang, K and Wang, Z},
title = {Efficient genetic transformation and genome editing via an Agrobacterium-mediated in commercial oat (Avena sativa L.) cultivars.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {7},
pages = {1697-1699},
doi = {10.1111/jipb.13915},
pmid = {40192038},
issn = {1744-7909},
support = {2005DKA21003//the National Center for Forestry and Grassland Genetic Resources/ ; },
mesh = {*Avena/genetics/microbiology ; *Gene Editing/methods ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; },
abstract = {An optimized Agrobacterium-mediated transformation protocol for immature and mature oat embryos increased transformation efficiencies and the number of transformable cultivars and enabled highly efficient CRISPR/Cas9 and CRISPR/Cas12i genome editing to accelerate oat biotechnology breeding.},
}

*Avena/genetics/microbiology
*Gene Editing/methods
*Transformation, Genetic
*Agrobacterium/genetics
Plants, Genetically Modified
CRISPR-Cas Systems/genetics
Genome, Plant/genetics

@article {pmid40187931,
year = {2025},
author = {Jiang, Y and Xiao, Z and Luo, Z and Zhou, S and Tong, C and Jin, S and Liu, X and Qin, R and Xu, R and Pan, L and Li, J and Wei, P},
title = {Improving plant C-to-G base editors with a cold-adapted glycosylase and TadA-8e variants.},
journal = {Trends in biotechnology},
volume = {43},
number = {7},
pages = {1765-1787},
doi = {10.1016/j.tibtech.2025.03.001},
pmid = {40187931},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; Oryza/genetics ; Plants, Genetically Modified/genetics ; *Cytidine Deaminase/genetics/metabolism ; *Uracil-DNA Glycosidase/genetics/metabolism ; Cytosine/metabolism ; Guanine/metabolism ; Humans ; CRISPR-Cas Systems ; Animals ; Cold Temperature ; },
abstract = {Plant cytosine (C)-to-guanine (G) base editors (CGBEs) have been established but suffer from limited editing efficiencies and low outcome purities. This study engineered a cod uracil DNA glycosylase (cod UNG, coUNG) from the cold-adapted fish Gadus morhua for plant CGBE, demonstrating 1.71- to 2.54-fold increases in C-to-G editing efficiency compared with the CGBE using human UNG (hUNG). Further engineering took advantage of TadA-8e-derived cytidine deaminases (TadA-CDs). These variants induced C substitutions with efficiencies ranging from 26.28% to 30.82% in rice cells, whereas adenine (A) conversion was negligible. By integrating coUNG and TadA-CDc elements with SpCas9 nickase, the resulting CDc-CGBEco achieved pure C-to-G editing without byproducts in up to 52.08% of transgenic lines. Whole-genome sequencing (WGS) analysis revealed no significant off-target effects of the CDc-BEs in rice. In addition, CDc-CGBEco enabled precise C-to-G editing in soybean and tobacco. These engineered CGBEs enhanced editing efficiency, purity, and specificity, suggesting their broad potential for applications in scientific research and crop breeding.},
}

*Gene Editing/methods
Oryza/genetics
Plants, Genetically Modified/genetics
*Cytidine Deaminase/genetics/metabolism
*Uracil-DNA Glycosidase/genetics/metabolism
Cytosine/metabolism
Guanine/metabolism
Humans
CRISPR-Cas Systems
Animals
Cold Temperature

@article {pmid40119518,
year = {2025},
author = {He, X and Yan, T and Song, Z and Xiang, L and Xiang, J and Yang, Y and Ren, K and Bu, J and Xu, X and Li, Z and Guo, X and Lin, B and Zhou, Q and Lin, G and Gu, F},
title = {Correcting a patient-specific Rhodopsin mutation with adenine base editor in a mouse model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {7},
pages = {3101-3113},
doi = {10.1016/j.ymthe.2025.03.021},
pmid = {40119518},
issn = {1525-0024},
mesh = {Animals ; Disease Models, Animal ; Mice ; *Gene Editing/methods ; *Adenine/metabolism ; Humans ; *Retinitis Pigmentosa/genetics/therapy ; *Mutation ; *Rhodopsin/genetics ; Gene Knock-In Techniques ; Dependovirus/genetics ; Genetic Vectors/genetics ; Electroretinography ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Retina/metabolism/pathology ; Genetic Therapy/methods ; },
abstract = {Genome editing offers a great promise to treating human genetic diseases. To assess genome-editing-mediated therapeutic effects in vivo, an animal model is indispensable. The genomic disparities between mice and humans often impede the direct clinical application of genome-editing-mediated treatments using conventional mouse models. Thus, the generation of a mouse model with a humanized genomic segment containing a patient-specific mutation is highly sought after for translational research. In this study, we successfully developed a knockin mouse model for autosomal-dominant retinitis pigmentosa (adRP), designated as hT17M knockin, which incorporates a 75-nucleotide DNA segment with the T17M mutation (Rhodopsin-c.C50T; p.T17M). This model demonstrated significant reductions in electroretinogram amplitudes and exhibited disruptions in retinal structure. Subsequently, we administered an adeno-associated virus vectors carrying an adenine base editor (ABE) and a single-guide RNA specifically targeting the T17M mutation, achieving a peak correction rate of 39.7% at the RNA level and significantly improving retinal function in ABE-injected mice. These findings underscore that the hT17M knockin mouse model recapitulates the clinical features of adRP patients and exhibits therapeutic effects with ABE-mediated treatments. It offers a promising avenue for the development of gene-editing therapies for RP.},
}

Animals
Disease Models, Animal
Mice
*Gene Editing/methods
*Adenine/metabolism
Humans
*Retinitis Pigmentosa/genetics/therapy
*Mutation
*Rhodopsin/genetics
Gene Knock-In Techniques
Dependovirus/genetics
Genetic Vectors/genetics
Electroretinography
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Retina/metabolism/pathology
Genetic Therapy/methods

@article {pmid38944889,
year = {2025},
author = {Kang, ES and Kim, NH and Lim, HK and Jeon, H and Han, K and No, YH and Kim, K and Khaleel, ZH and Shin, D and Eom, K and Nam, J and Lee, BS and Kim, HJ and Suh, M and Lee, J and Thach, TT and Hyun, J and Kim, YH},
title = {Structure-Guided Engineering of Thermodynamically Enhanced SaCas9 for Improved Gene Suppression.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {26},
pages = {e2404680},
doi = {10.1002/adma.202404680},
pmid = {38944889},
issn = {1521-4095},
support = {//National Research Foundation of Korea/ ; NRF-2023R1A2C3005731//Korean government/ ; RS-2023-00302458)//Korean government/ ; IBS-R015-D1//Institute for Basic Science/ ; },
mesh = {Animals ; Mice ; *Protein Engineering/methods ; Thermodynamics ; Humans ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; DNA/metabolism ; },
abstract = {Proteins with multiple domains play pivotal roles in various biological processes, necessitating a thorough understanding of their structural stability and functional interplay. Here, a structure-guided protein engineering approach is proposed to develop thermostable Cas9 (CRISPR-associated protein 9) variant for CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference applications. By employing thermodynamic analysis, combining distance mapping and molecular dynamics simulations, deletable domains are identified to enhance stability while preserving the DNA recognition function of Cas9. The resulting engineered Cas9, termed small and dead form Cas9, exhibits improved thermostability and maintains target DNA recognition function. Cryo-electron microscopy analysis reveals structural integrity with reduced atomic density in the deleted domain. Fusion with functional elements enables intracellular delivery and nuclear localization, demonstrating efficient gene suppression in diverse cell types. Direct delivery in the mouse brain shows enhanced knockdown efficiency, highlighting the potential of structure-guided engineering to develop functional CRISPR systems tailored for specific applications. This study underscores the significance of integrating computational and experimental approaches for protein engineering, offering insights into designing tailored molecular tools for precise biological interventions.},
}

Animals
Mice
*Protein Engineering/methods
Thermodynamics
Humans
Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
CRISPR-Cas Systems
HEK293 Cells
DNA/metabolism

@article {pmid40612691,
year = {2024},
author = {Lin, J and Yang, J},
title = {CRISPR-Cas systems: A revolution in genome editing and its diverse applications.},
journal = {Journal of biomed research},
volume = {5},
number = {1},
pages = {108-114},
pmid = {40612691},
issn = {2693-5910},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) Cas (CRISPR6 associated protein) system is an advanced adaptive immune system found in prokaryotes. First discovered in1987, CRISPR Cas has revolutionized genetic research in the past two decades. CRISPR-Cas9 the most widespread system enables precise gene editing by creating double strand breaks. Its ease of use and cost-effectiveness has lowered the barrier to entry for genetic research. CRISPR holds immense potential in many fields from agriculture to medicine. In agriculture, CRISPR has accelerated crop improvement by enabling precise gene edits for desirable traits. In medicine, CRISPR holds promise in xenotransplant, cancers and infectious diseases (HIV) treatment. This review traces the historical development of CRISPR-Cas systems, explores their unique applications, and discusses future advancements aimed at enhancing CRISPR's precision and expanding its applications through technologies like prime and base editing.},
}

@article {pmid40603034,
year = {2025},
author = {Sugo, T and Shirasago, Y and Yoshimoto, S and Kitajima, M},
title = {[GenAhead Bio: your partner for extensive support of genome editing and co-‍development of nucleic acid delivery].},
journal = {Nihon yakurigaku zasshi. Folia pharmacologica Japonica},
volume = {160},
number = {4},
pages = {274-278},
doi = {10.1254/fpj.25004},
pmid = {40603034},
issn = {0015-5691},
mesh = {*Gene Editing ; Humans ; *Nucleic Acids/administration & dosage ; CRISPR-Cas Systems ; Animals ; RNA, Small Interfering ; },
abstract = {Inspired by my experiences working in research at an overseas biotech venture, I founded GenAhead Bio Inc. in 2018. GenAhead Bio adopts a unique dual-business structure, providing contract services for generating genetically modified cells using highly efficient CRISPR/Cas9 genome editing technology for researchers, while simultaneously pursuing a nucleic acid drug business aiming to develop nucleic acid drugs such as antisense oligonucleotides and siRNAs. Based on the emerging delivery system called Antibody-Nucleic acid Conjugate, where an antibody is covalently linked to a nucleic acid as a targeting ligand, we are conducting drug developmental research by delivering nucleic acids to the organs where antibodies accumulate. Our ultimate goal is to apply this technology to genome editing for gene modification in specific cell types. In this review, we will introduce some case studies of genome editing, including single nucleotide substitutions, as well as the delivery of siRNA to the skeletal muscle using anti-transferrin receptor (CD71) antibody and its therapeutic effects on muscular diseases.},
}

*Gene Editing
Humans
*Nucleic Acids/administration & dosage
CRISPR-Cas Systems
Animals
RNA, Small Interfering

@article {pmid40601773,
year = {2025},
author = {Chafe, SC and Zhai, K and Aghaei, N and Miletic, P and Huang, Z and Brown, KR and Mobilio, D and Young, D and Suk, Y and Grewal, S and McKenna, D and Alizada, Z and Kieliszek, AM and Lam, FC and Escudero, L and Huang, Q and Huebner, A and Lu, J and Ang, P and Anand, A and Custers, S and Apel, E and Slassi, S and Brakel, B and Kim, J and Liu, JKC and Bassey-Archibong, BI and Abdo, R and Shargall, Y and Lu, JQ and Cutz, JC and Zhang, Q and Li, SS and Venugopal, C and Hynds, RE and Dufour, A and Moffat, J and Swanton, C and Bao, S and Singh, SK},
title = {A genome-wide in vivo CRISPR activation screen identifies BACE1 as a therapeutic vulnerability of lung cancer brain metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {805},
pages = {eadu2459},
doi = {10.1126/scitranslmed.adu2459},
pmid = {40601773},
issn = {1946-6242},
mesh = {Humans ; *Brain Neoplasms/secondary/genetics ; *Lung Neoplasms/pathology/genetics ; *Amyloid Precursor Protein Secretases/metabolism/genetics ; *Aspartic Acid Endopeptidases/metabolism/genetics ; Animals ; *Carcinoma, Non-Small-Cell Lung/genetics/pathology ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice ; ErbB Receptors/metabolism ; *CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Expression Regulation, Neoplastic ; },
abstract = {Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation screen to identify molecular drivers of brain metastasis from an orthotopic xenograft model derived from a patient with NSCLC. We found that activating expression of the Alzheimer's disease-associated beta-secretase 1 (BACE1) led to a substantial increase in brain metastases. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified that BACE1 acts through epidermal growth factor receptor to drive this metastatic phenotype. Together, our data highlight the power of in vivo CRISPR activation screening to unveil molecular drivers and potential therapeutic targets of NSCLC brain metastasis.},
}

Humans
*Brain Neoplasms/secondary/genetics
*Lung Neoplasms/pathology/genetics
*Amyloid Precursor Protein Secretases/metabolism/genetics
*Aspartic Acid Endopeptidases/metabolism/genetics
Animals
*Carcinoma, Non-Small-Cell Lung/genetics/pathology
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice
ErbB Receptors/metabolism
*CRISPR-Cas Systems/genetics
Xenograft Model Antitumor Assays
Gene Expression Regulation, Neoplastic

@article {pmid40599263,
year = {2025},
author = {Yang, R and Tang, T and Wulae, and Liu, P and Wang, X and Jiang, Y and Zhang, S and Wang, M and Yang, L and Wang, C},
title = {[One-Step Detection of Human Influenza B Virus Through Recombinase Polymerase Amplification and CRISPR/Cas12a Protein].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {2},
pages = {549-555},
pmid = {40599263},
issn = {1672-173X},
mesh = {*Influenza B virus/isolation & purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Recombinases/genetics/metabolism ; Sensitivity and Specificity ; *Influenza, Human/diagnosis/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: To establish a one-step detection method based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a protein for the rapid and sensitive detection of human influenza B virus.

METHODS: RPA amplification primers were designed according to the conserved gene (NS1 gene) of human influenza B virus (Victoria lineage). The reaction system was established using the standard plasmid as the template. First of all, the reaction system was incubated at 37 ℃ for 15 minutes for RPA amplification. Then, the CRISPR/Cas12a system on the tube cap was thoroughly mixed with the RPA amplification product at the bottom of the tube through fast centrifugation, and real-time fluorescence detection was carried out at 37 ℃. The reaction conditions were optimized to establish a one-step RPA-CRISPR/Cas12a detection method for human influenza B virus. The sensitivity of the testing method was evaluated using standard plasmids and pseudoviruses, and the specificity was evaluated using other viruses that may cause febrile respiratory syndrome. The consistency between the results of the one-step detection method and those of RT-qPCR detection was evaluated by testing real samples.

RESULTS: A one-step detection method based on RPA-CRISPR/Cas12a was successfully established. The optimal reaction conditions included a reaction temperature of 37 ℃, a Cas12a/crRNA concentation ratio of 1∶1, a Cas12a concentration of 120 nmol/L, a single-stranded DNA (ssDNA) probe concentration of 300 nmol/L, and a primer concentration of 480 nmol/L. The method could detect standard plasmid DNA as low as 2.8 copies/μL within 25 minutes and pseudoviruses as low as 2.77 copies/μL within 30 minutes. The testing method showed high specificity, and no cross-reaction was observed with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A (H1N1) virus, or respiratory syncytial virus subgroup A. When testing clinical samples, the sensitivity and the specificity for examining clinical samples were 93.33% and 100%, respectively, and consistency with RT-qPCR results was 97.14%.

CONCLUSION: With the one-step detection method based on RPA-CRISPR/Cas12a established in this study, the whole sample detection process, including nucleic acid release, reverse transcription, isothermal amplification, CRISPR/Cas12a system cleavage, and fluorescence signal output, can be completed within 30 minutes. Its high sensitivity, specificity, and successful application in clinical samples highlight its potential for rapid point-of-care testing in clinical settings.},
}

*Influenza B virus/isolation & purification/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Recombinases/genetics/metabolism
Sensitivity and Specificity
*Influenza, Human/diagnosis/virology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40598996,
year = {2025},
author = {Han, HG and Nandre, R and Eom, H and Choi, YJ and Ro, HS},
title = {Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {6},
pages = {e2502011},
doi = {10.71150/jm.2502011},
pmid = {40598996},
issn = {1976-3794},
support = {2023R1A2C1007213//National Research Foundation of Korea/ ; //Ministry of Science and ICT/ ; RS2024-00322425//Rural Development Administration/ ; },
mesh = {*CRISPR-Cas Systems ; *Paecilomyces/genetics/metabolism ; Promoter Regions, Genetic ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics/metabolism ; Fungal Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (PH4, PP2, PS8, Ptub, Ptef1, and PgpdA), along with a signal sequence-tagged eGFP, flanked by 5'-upstream (336 bp) and 3'-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that PgpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting PgpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.},
}

*CRISPR-Cas Systems
*Paecilomyces/genetics/metabolism
Promoter Regions, Genetic
*Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
*Genetic Engineering/methods
Green Fluorescent Proteins/genetics/metabolism
Fungal Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40598890,
year = {2025},
author = {Wei, W and Gao, CH and Jiang, X and Qiao, J and Zhang, L and Yan, Y and Zhao, G and Yang, K and Yan, J and Yang, M},
title = {CARF-dependent preferential RNA cleavage by Csm6 increases drug susceptibility of mycobacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf622},
pmid = {40598890},
issn = {1362-4962},
support = {32070079//National Natural Science Foundation of China/ ; 32370127//National Natural Science Foundation of China/ ; 2662025ZHPY001//Central Universities/ ; 2020kfyXJJS119//Central Universities/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mycobacterium smegmatis/genetics/drug effects/metabolism ; *RNA Cleavage ; *Mycobacterium tuberculosis/genetics/drug effects ; *Bacterial Proteins/genetics/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Mycolic Acids/metabolism ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that defend against invading mobile genetic elements. The type III-A CRISPR-Cas system has been studied in the evolutionary and epidemiological context of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, its biological function remains poorly understood. Here, we demonstrate that heterologous expression of csm6, a single-stranded RNA ribonuclease of the CRISPR-Cas system, exhibits preferential RNA cleavage activity targeting host transcripts. This activity significantly downregulates ribosomal and mycolic acid biosynthesis pathway genes, leading to a global reduction in translation levels and an increased drug susceptibility of Mycobacterium smegmatis. Furthermore, mutagenesis analysis revealed that Csm6's biological function critically depends on its CARF domain rather than its HEPN domain. In conclusion, our study elucidates the biological role of the Csm6 protein in the CRISPR-Cas system, both in vitro and in vivo, highlighting how preferential RNA cleavage impacts multiple mycobacterial processes. These findings provide novel insights into the functional diversity of CRISPR-Cas systems in mycobacteria.},
}

*CRISPR-Cas Systems/genetics
*Mycobacterium smegmatis/genetics/drug effects/metabolism
*RNA Cleavage
*Mycobacterium tuberculosis/genetics/drug effects
*Bacterial Proteins/genetics/metabolism/chemistry
Drug Resistance, Bacterial/genetics
Mycolic Acids/metabolism
Gene Expression Regulation, Bacterial

@article {pmid40598087,
year = {2025},
author = {Garimella, SS and Minami, SA and Khanchandani, AN and Abad Santos, JC and Schaffer, SR and Shah, PS},
title = {A simplified two-plasmid system for orthogonal control of mammalian gene expression using light-activated CRISPR effector.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {58},
pmid = {40598087},
issn = {1472-6750},
support = {n/a//AMPAC Fine Chemicals Summer Research Internship/ ; n/a//Hellman Family Foundation/ ; n/a//Good Food Institute/ ; },
mesh = {*Plasmids/genetics ; HEK293 Cells ; Humans ; *Optogenetics/methods ; Green Fluorescent Proteins/genetics/metabolism ; Light ; *CRISPR-Cas Systems/genetics ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression Regulation ; Gene Expression ; },
abstract = {BACKGROUND: Optogenetic systems use light-responsive proteins to control gene expression, ion channels, protein localization, and signaling with the "flip of a switch". One such tool is the light activated CRISPR effector (LACE) system. Its ability to regulate gene expression in a tunable, reversible, and spatially resolved manner makes it attractive for many applications. However, LACE relies on delivery of four separate components on individual plasmids, which can limit its use. Here, we optimize LACE to reduce the number of plasmids needed to deliver all four components.

RESULTS: The two-plasmid LACE (2pLACE) system combines the four components of the original LACE system into two plasmids. Following construction, the behavior of 2pLACE was rigorously tested using optogenetic control of enhanced green fluorescent protein (eGFP) expression as a reporter. Using human HEK293T cells, we optimized the ratio of the two plasmids, measured activation as a function of light intensity, and determined the frequency of the light to activate the maximum fluorescence. Overall, the 2pLACE system showed a similar dynamic range, tunability, and activation kinetics as the original four plasmid LACE (4pLACE) system. Interestingly, 2pLACE also had less variability in activation signal compared to 4pLACE. We also demonstrate the optimal LACE system also depends on cell type. In mouse myoblast C2C12 cells, 2pLACE displayed less variability compared to 4pLACE, similar to HEK293T cells. However, 2pLACE also had a smaller dynamic range in C2C12 cells compared to 4pLACE.

CONCLUSIONS: This simplified system for optogenetics will be more amenable to biotechnology applications where variability needs to be minimized. By optimizing the LACE system to use fewer plasmids, 2pLACE becomes a flexible tool in multiple research applications. However, the optimal system may depend on cell type and application.},
}

*Plasmids/genetics
HEK293 Cells
Humans
*Optogenetics/methods
Green Fluorescent Proteins/genetics/metabolism
Light
*CRISPR-Cas Systems/genetics
Animals
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Expression Regulation
Gene Expression

@article {pmid40598062,
year = {2025},
author = {Ajdanian, L and Torkamaneh, D},
title = {Mother transformer: A High-Throughput, Cost-Effective in Planta Hairy Root Transformation Method for Cannabis.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {60},
pmid = {40598062},
issn = {1472-6750},
mesh = {*Plant Roots/genetics/microbiology ; *Plants, Genetically Modified/genetics ; *Agrobacterium/genetics ; *Transformation, Genetic ; *Cannabis/genetics ; Cost-Benefit Analysis ; *High-Throughput Screening Assays/methods ; },
abstract = {BACKGROUND: Hairy root (HR) transformation assays mediated by Agrobacterium rhizogenes, both in vitro and ex vitro, are essential tools in plant biotechnology and functional genomics. These assays can be significantly influenced by various factors, which ultimately can enhance the efficiency. In this study, we optimized a two-step ex vitro HR transformation method using the actual mother plant combined with the RUBY system and compared with existing methods.

RESULTS: The two-step ex vitro method proved more efficient than both the one-step ex vitro and in vitro methods, with the highest transformation efficiency of 90% observed in the actual plant. This technique also demonstrated a faster and less complicated approach, reducing time to achieve massive transgenic HR formation by 9-29 days compared to other methods.

CONCLUSIONS: A novel, quicker, less complicated, and more efficient two-step transformation method for cannabis has been established, presenting a significantly lower risk of contamination. This protocol is particularly interesting to produce secondary metabolites using the CRISPR/Cas system in cannabis. We anticipate that this method will facilitate substantial time savings by rapidly producing hundreds of transformed samples.},
}

*Plant Roots/genetics/microbiology
*Plants, Genetically Modified/genetics
*Agrobacterium/genetics
*Transformation, Genetic
*Cannabis/genetics
Cost-Benefit Analysis
*High-Throughput Screening Assays/methods

@article {pmid40597657,
year = {2025},
author = {Malekos, E and Montano, C and Carpenter, S},
title = {CRISPRware: a software package for contextual gRNA library design.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {607},
pmid = {40597657},
issn = {1471-2164},
support = {F31AI179201//National Institute of Allergy and Infectious Diseases/ ; R35GM137801/GM/NIGMS NIH HHS/United States ; },
mesh = {*Software ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; *CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; Humans ; Animals ; },
abstract = {We present CRISPRware, an efficient method for generating guide RNA (gRNA) libraries against transcribed, translated, and noncoding regions. CRISPRware leverages next-generation sequencing data to design context-specific gRNAs and can account for genetic variation, which allows allele-specific guide design on a genome-wide scale. As a demonstration of use and to create a publicly available resource, we use CRISPRware to identify and score gRNAs against coding sequences in six model organisms for Cas9 and Cas12A and host these in a publicly available session on the UCSC Genome Browser.},
}

*Software
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Library
*CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing
Humans
Animals

@article {pmid40596971,
year = {2025},
author = {Martí-Díaz, R and Sánchez-Del-Campo, L and Montenegro, MF and Hernández-Caselles, T and Piñero-Madrona, A and Cabezas-Herrera, J and Rodríguez-López, JN},
title = {Ex vivo engineering of phagocytic signals in breast cancer cells for a whole tumor cell-based vaccine.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1029},
pmid = {40596971},
issn = {1471-2407},
support = {21407/FPI/20//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; FSRM/10.13039/100007801(22544/PI/24)//Fundación Séneca/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; CPP2023-010510//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; PID2023-149281OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; },
mesh = {Animals ; Female ; Mice ; *Phagocytosis/immunology ; *Cancer Vaccines/immunology ; *Breast Neoplasms/immunology/therapy/pathology ; Humans ; CD47 Antigen/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; Macrophages/immunology ; Mice, Inbred BALB C ; },
abstract = {BACKGROUND: Today, cell therapies are constantly evolving and providing new options for cancer patients. These therapies are mostly based on the inoculation of immune cells extracted from a person's own tumor; however, some studies using whole tumor cell-based vaccines are approaching the level of maturity required for clinical use. Although these latest therapies will have to be developed further and adapted to overcome many ethical barriers, there is no doubt that therapeutic cancer vaccines are the next frontier of immunotherapy.

METHODS: Ionizing radiation and CD47 knockout via CRISPR-Cas9 genome editing were used to optimize the macrophage-mediated phagocytosis of breast cancer cells. These cells were subsequently used in several mouse models to determine their potential as novel whole-cell-based vaccines to drive antitumor immunity. To improve the recognition of tumor cells by activated immune cells, this cellular therapy was combined with anti-PD-1 antibody treatments.

RESULTS: Here, we showed that irradiation of 4T1 breast cancer cells increases their immunogenicity and, when injected into the blood of immunocompetent mice, elicits a complete antitumor immune response mediated, in part, by the adaptive immune system. Next, to improve the macrophage-mediated phagocytosis of breast cancer cells, we knocked out CD47 in 4T1 cells. When injected in the bloodstream, irradiated CD47 knockout cells activated both the adaptive and the innate immune systems. Therefore, we used these ex vivo engineered cells as a whole tumor cell-based vaccine to treat breast tumors in immunocompetent mice. A better response was obtained when these cells were combined with an anti-PD-1 antibody.

CONCLUSION: These results suggest that tumor cells obtained from surgical samples of a breast cancer patient could be engineered ex vivo and used as a novel cell therapy to drive antitumor immunity.},
}

Animals
Female
Mice
*Phagocytosis/immunology
*Cancer Vaccines/immunology
*Breast Neoplasms/immunology/therapy/pathology
Humans
CD47 Antigen/genetics
Cell Line, Tumor
CRISPR-Cas Systems
Macrophages/immunology
Mice, Inbred BALB C

@article {pmid40596718,
year = {2025},
author = {Schuster, B and Dobiášovská, I and Ćurčić, J and Pajer, P and Borna, Š and Bartůněk, P},
title = {SWITCHER, a CRISPR-inducible floxed wild-type Cre regulating CRISPR activity.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {982},
pmid = {40596718},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; *Integrases/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Humans ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacterial Proteins ; },
abstract = {Although several Cre-regulated CRISPR/Cas platforms exist, a CRISPR/Cas-controlled Cre-system remains a challenge. Here, we present a genetic switch we term SWITCHER based on a floxed wild-type Cre-construct representing a CRISPR-inducible and self-limiting kill switch. By leveraging CRISPR/Cas12a-mediated crRNA-array maturation, we showcase SWITCHER's dual role-not just as a recombinase but as a CRISPR switch, capable of orchestrating distinct Cas12a/crRNA-encoded programs.},
}

*CRISPR-Cas Systems/genetics
*Integrases/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing/methods
Humans
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics
Bacterial Proteins

@article {pmid40595661,
year = {2025},
author = {Boswell, CW and Hoppe, C and Sherrard, A and Miao, L and Kojima, ML and Martino, P and Zhao, N and Stasevich, TJ and Nicoli, S and Giraldez, AJ},
title = {Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5503},
pmid = {40595661},
issn = {2041-1723},
support = {R01 HD100035/HD/NICHD NIH HHS/United States ; R35 GM122580/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems/genetics ; Zebrafish Proteins/metabolism/genetics ; Single-Domain Antibodies/genetics/metabolism ; Epitopes/genetics/metabolism ; Gene Knock-In Techniques/methods ; Humans ; Single-Chain Antibodies/genetics/metabolism ; },
abstract = {Probing endogenous protein localization and function in vivo remains challenging due to laborious gene targeting and monofunctional alleles. Here, we develop a multifunctional and adaptable toolkit based on genetically encoded affinity reagents (GEARs). GEARs use small epitopes recognized by nanobodies and single chain variable fragments to enable fluorescent visualization, manipulation and degradation of protein targets in vivo. Furthermore, we outline a CRISPR/Cas9-based epitope tagging pipeline to demonstrate its utility for producing knock-in alleles that have broad applications. We use GEARs to examine the native behavior of the pioneer transcription factor Nanog and the planar cell polarity protein Vangl2 during early zebrafish development. Together, this toolkit provides a versatile system for probing and perturbing endogenous protein function while circumventing challenges associated with conventional gene targeting and is broadly available to the model organism community.},
}

Animals
Zebrafish/genetics/embryology/metabolism
CRISPR-Cas Systems/genetics
Zebrafish Proteins/metabolism/genetics
Single-Domain Antibodies/genetics/metabolism
Epitopes/genetics/metabolism
Gene Knock-In Techniques/methods
Humans
Single-Chain Antibodies/genetics/metabolism

@article {pmid40595633,
year = {2025},
author = {Fu, W and Ma, J and Wang, Z and Tang, N and Pan, D and Su, M and Wu, Z and Gan, J and Ji, Q},
title = {Mechanisms and engineering of a miniature type V-N CRISPR-Cas12 effector enzyme.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5667},
pmid = {40595633},
issn = {2041-1723},
support = {22277078//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure ; Humans ; DNA/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure ; Protein Engineering ; },
abstract = {Type V CRISPR-Cas12 systems are highly diverse in their functionality and molecular compositions, including miniature Cas12f1 and Cas12n genome editors that provide advantages for efficient in vivo therapeutic delivery due to their small size. In contrast to Cas12f1 nucleases that utilize a homodimer structure for DNA targeting and cleavage with a preference for T- or C-rich PAMs, Cas12n nucleases are likely monomeric proteins and uniquely recognize rare A-rich PAMs. However, the molecular mechanisms behind RNA-guided genome targeting and cleavage by Cas12n remain unclear. Here, we present the cryo-electron microscopy (cryo-EM) structure of Rothia dentocariosa Cas12n (RdCas12n) bound to a single guide RNA (sgRNA) and target DNA, illuminating the intricate molecular architecture of Cas12n and its sgRNA, as well as PAM recognition and nucleic-acid binding mechanisms. Through structural comparisons with other Cas12 nucleases and the ancestral precursor TnpB, we provide insights into the evolutionary significance of Cas12n in the progression from TnpB to various Cas12 nucleases. Additionally, we extensively modify the sgRNA and convert RdCas12n into an effective genome editor in human cells. Our findings enhance the understanding of the evolutionary mechanisms of type V CRISPR-Cas12 systems and offer a molecular foundation for engineering Cas12n genome editors.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Cryoelectron Microscopy
Gene Editing/methods
*CRISPR-Associated Proteins/metabolism/genetics/chemistry/ultrastructure
Humans
DNA/metabolism/genetics
*Bacterial Proteins/metabolism/genetics/chemistry/ultrastructure
Protein Engineering

@article {pmid40595632,
year = {2025},
author = {Zhang, Y and Zhang, T and Xiao, X and Wang, Y and Kawalek, A and Ou, J and Ren, A and Sun, W and de Bakker, V and Liu, Y and Li, Y and Yang, L and Ye, L and Jia, N and Veening, JW and Liu, X},
title = {CRISPRi screen identifies FprB as a synergistic target for gallium therapy in Pseudomonas aeruginosa.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5870},
pmid = {40595632},
issn = {2041-1723},
support = {82270012//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82200047//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Pseudomonas aeruginosa/drug effects/genetics ; *Gallium/pharmacology/therapeutic use ; Animals ; *Pseudomonas Infections/drug therapy/microbiology ; Mice ; Biofilms/drug effects/growth & development ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; Reactive Oxygen Species/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxidative Stress/drug effects ; CRISPR-Cas Systems ; Female ; Iron/metabolism ; },
abstract = {With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB, encoding a ferredoxin-NADP[+] reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium's efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa, suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa, advancing the identification of novel therapeutic targets.},
}

*Pseudomonas aeruginosa/drug effects/genetics
*Gallium/pharmacology/therapeutic use
Animals
*Pseudomonas Infections/drug therapy/microbiology
Mice
Biofilms/drug effects/growth & development
*Anti-Bacterial Agents/pharmacology
Humans
Microbial Sensitivity Tests
Reactive Oxygen Species/metabolism
*Bacterial Proteins/genetics/metabolism
Oxidative Stress/drug effects
CRISPR-Cas Systems
Female
Iron/metabolism

@article {pmid40595611,
year = {2025},
author = {Irie, N and Takeda, N and Satou, Y and Araki, K and Ono, M},
title = {Machine learning-assisted decoding of temporal transcriptional dynamics via fluorescent timer.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5720},
pmid = {40595611},
issn = {2041-1723},
support = {DCRPGF\100007//Cancer Research UK (CRUK)/ ; JP21K07082, JP21H00433, JP24K10259, JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPJSCCA2020008//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16H0627601, JP22H04922//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24gm1810001s0103//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; *Machine Learning ; *Forkhead Transcription Factors/genetics/metabolism ; Mice ; *Transcription, Genetic ; Flow Cytometry/methods ; Gene Expression Regulation ; Mice, Inbred C57BL ; Single-Cell Analysis/methods ; Enhancer Elements, Genetic ; T-Lymphocytes, Regulatory/metabolism ; Genes, Reporter ; Neural Networks, Computer ; Spleen/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Investigating the temporal dynamics of gene expression is crucial for understanding gene regulation across various biological processes. Using the Fluorescent Timer protein, the Timer-of-cell-kinetics-and-activity system enables analysis of transcriptional dynamics at the single-cell level. However, the complexity of Timer fluorescence data has limited its broader application. Here, we introduce an integrative approach combining molecular biology and machine learning to elucidate Foxp3 transcriptional dynamics through flow cytometric Timer analysis. We have developed a convolutional neural network-based method that incorporates image conversion and class-specific feature visualisation for class-specific feature identification at the single-cell level. Biologically, we developed a novel CRISPR mutant of Foxp3 fluorescent Timer reporter mice lacking the enhancer Conserved Non-coding Sequence 2, which revealed new roles of this enhancer in regulating Foxp3 transcription frequency under specific conditions. Furthermore, analysis of wild-type Foxp3 fluorescent Timer reporter mice at different ages uncovered distinct patterns of Foxp3 expression from neonatal to aged mice, highlighting prominent thymus-like features of neonatal splenic Foxp3[+] T cells. In conclusion, our study uncovers previously unrecognised Foxp3 transcriptional dynamics, establishing a proof-of-concept for integrating CRISPR, single-cell dynamics analysis, and machine learning methods as advanced techniques to understand transcriptional dynamics in vivo.},
}

Animals
*Machine Learning
*Forkhead Transcription Factors/genetics/metabolism
Mice
*Transcription, Genetic
Flow Cytometry/methods
Gene Expression Regulation
Mice, Inbred C57BL
Single-Cell Analysis/methods
Enhancer Elements, Genetic
T-Lymphocytes, Regulatory/metabolism
Genes, Reporter
Neural Networks, Computer
Spleen/metabolism/cytology
CRISPR-Cas Systems

@article {pmid40595537,
year = {2025},
author = {Hofmann, R and Herman, C and Mo, CY and Mathai, J and Marraffini, LA},
title = {Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5730},
pmid = {40595537},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Bacterial Proteins/genetics/metabolism ; Bacteriophages/genetics ; *Endonucleases/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.},
}

*CRISPR-Cas Systems/genetics
*Streptococcus pyogenes/genetics/virology
*CRISPR-Associated Proteins/genetics/metabolism
Mutation
*Bacterial Proteins/genetics/metabolism
Bacteriophages/genetics
*Endonucleases/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40595093,
year = {2025},
author = {Wei, T and Yan, Y and Niu, M and Dong, X and Li, H and Sun, Y and Fa, Y},
title = {A rapid LASV detection method based on CRISPR-Cas13a and recombinase aided amplification with special lateral-flow test strips.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {20640},
pmid = {40595093},
issn = {2045-2322},
support = {2023YFC2605100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Lassa virus/isolation & purification/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *Lassa Fever/diagnosis/virology ; Reagent Strips ; Animals ; },
abstract = {Lassa virus (LASV) is a high-risk pathogen associated with severe viral hemorrhagic fever in both humans and animals. Owing to its significant treatment challenges and high infectivity, LASV is classified as a biosafety level 4 (BSL-4) pathogen. It is essential to establish a rapid LASV detection method to prevent and control the disease. To address the biosecurity threats caused by LASV, in this study, we developed a new test method for LASV detection by combining the recombinase-mediated isothermal amplification (RAA) and CRISPR-Cas13a detection technology. The detection efficiency of this method was evaluated and compared with existing methods. The results demonstrate that this new detection maintains relatively high sensitivity and specificity, while having excellent simplicity and rapidity. The sensitivity of the method for detecting the LASV can achieve a threshold of 10[1] copies/µL using fluorescence detection in 90 min and 10[2] copies/µL with lateral flow strip detection in just an hour, which only needs a simple constant temperature equipment to achieve. The application of this detection method holds substantial biosecurity significance for underdeveloped regions (e.g., West Africa), as well as for countries like China, which have a vast territory and uneven development of medical testing levels in various regions.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Lassa virus/isolation & purification/genetics
Humans
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*Lassa Fever/diagnosis/virology
Reagent Strips
Animals

@article {pmid40594904,
year = {2025},
author = {Sayem, M and Rafi, MA and Mishu, ID and Mahmud, Z},
title = {Comprehensive genomic analysis reveals virulence and antibiotic resistance genes in a multidrug-resistant Bacillus cereus isolated from hospital wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {22915},
pmid = {40594904},
issn = {2045-2322},
mesh = {*Bacillus cereus/genetics/pathogenicity/isolation & purification/drug effects ; *Wastewater/microbiology ; Bangladesh ; *Drug Resistance, Multiple, Bacterial/genetics ; Phylogeny ; Hospitals ; Virulence/genetics ; Genome, Bacterial ; Whole Genome Sequencing ; Genomics/methods ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Humans ; },
abstract = {Hospital wastewater represents a significant reservoir for antimicrobial-resistant bacteria, including multidrug-resistant (MDR) Bacillus cereus, a pathogen of growing concern due to its potential impact on public health and environmental safety. This study characterizes the genomic features, antimicrobial resistance (AMR) mechanisms, and virulence potential of Bacillus cereus MBC, isolated from hospital wastewater in Dhaka, Bangladesh. Using whole-genome sequencing (WGS) and advanced bioinformatics, we analyzed the isolate's taxonomy, phylogenetics, functional annotation, and biosynthetic potential. The genome, spanning 5.6 Mb with a GC content of 34.84%, contained 5,881 protein-coding sequences, including 1,424 hypothetical proteins, and 28 genes associated with AMR. Phylogenetic analysis revealed a close genetic relationship with Bacillus cereus ATCC 14579, sharing virulence factors such as hemolysin BL (HBL), non-hemolytic enterotoxin (NHE), and cytotoxin K (CytK), all contributing to its pathogenicity. The ability to form biofilms further enhances the strain's persistence and resistance in hospital environments. AMR profiling identified genes conferring resistance to beta-lactams (e.g., BcI, BcII, BcIII), tetracyclines (tetB(P)), glycopeptides (vanY), and fosfomycin, highlighting the bacterium's capacity to resist a wide array of antibiotics. Functional annotation revealed metabolic pathways involved in iron acquisition and the biosynthesis of siderophores such as petrobactin and bacillibactin, reinforcing the bacterium's adaptability in nutrient-limited environments. Mobile genetic elements, including prophages, CRISPR-Cas systems, and transposable elements, suggest significant horizontal gene transfer (HGT), enhancing genetic plasticity and resistance spread. Pangenomic analysis, involving 125 B. cereus strains, revealed a high degree of genetic diversity and close relationships with strains from clinical, food, and agricultural environments, emphasizing the overlap between clinical and environmental reservoirs of resistance. The strain's isolation from hospital wastewater underscores the complex interplay between environmental contaminants and bacterial evolution, which fosters MDR traits. Our findings underscore the urgent need for enhanced genomic surveillance and wastewater management strategies to mitigate the spread of MDR B. cereus and AMR genes in hospital environments.},
}

*Bacillus cereus/genetics/pathogenicity/isolation & purification/drug effects
*Wastewater/microbiology
Bangladesh
*Drug Resistance, Multiple, Bacterial/genetics
Phylogeny
Hospitals
Virulence/genetics
Genome, Bacterial
Whole Genome Sequencing
Genomics/methods
Anti-Bacterial Agents/pharmacology
Virulence Factors/genetics
Humans

@article {pmid40594722,
year = {2025},
author = {Ren, X and Yao, XR and Chen, K and Xiao, WT and He, JY},
title = {CRISPR-Cas9 screening identifies a gene signature predictive of prognosis in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {21077},
pmid = {40594722},
issn = {2045-2322},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *CRISPR-Cas Systems/genetics ; *Brain Neoplasms/genetics/pathology/mortality ; Gene Expression Regulation, Neoplastic ; Biomarkers, Tumor/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; *Transcriptome ; },
abstract = {Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by a poor prognosis and limited therapeutic options. Identifying essential genes and pathways involved in GBM proliferation is important for developing prognostic biomarkers and potential therapeutic targets. In this study, genome-wide CRISPR-Cas9 screening data from the dependency map (DepMap) database were analyzed to explore proliferation-related essential genes and pathways in GBM. A five-gene prognostic signature-CLSPN, HSP90B1, MED10, SAMM50, and TOMM20-was constructed using univariate, LASSO, and multivariate Cox regression analyses, and its prognostic value was evaluated in independent cohorts. Weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA) suggested that the E2F targets pathway may be involved in GBM proliferation, consistent with the CRISPR screening results. Among the identified genes, MED10 was preliminarily implicated in regulating GBM cell proliferation and migration, as supported by functional assays. These findings propose a proliferation-related gene signature with potential prognostic relevance in GBM and indicate the E2F targets pathway as a biological process potentially associated with tumor progression. MED10 warrants further investigation as a candidate gene in the context of GBM biology and therapy.},
}

Humans
*Glioblastoma/genetics/pathology/mortality
Prognosis
*CRISPR-Cas Systems/genetics
*Brain Neoplasms/genetics/pathology/mortality
Gene Expression Regulation, Neoplastic
Biomarkers, Tumor/genetics
Cell Proliferation/genetics
Gene Expression Profiling
Gene Regulatory Networks
*Transcriptome

@article {pmid40593724,
year = {2025},
author = {Hu, M and Zhang, B and Shan, Y and Cao, F and Wang, Y and Qi, W and Wang, X and Shen, Y and Guo, X and Zhang, M and Tian, T and Xie, W and Zhang, M and Liang, F and Pei, D and Zhou, X},
title = {Scalable modulation of CRISPR‒Cas enzyme activity using photocleavable phosphorothioate DNA.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5939},
pmid = {40593724},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32300692//National Natural Science Foundation of China (National Science Foundation of China)/ ; 92068201//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M760985//China Postdoctoral Science Foundation/ ; GZB20240235//China Postdoctoral Science Foundation/ ; 2024T170292//China Postdoctoral Science Foundation/ ; 2023M741235//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics ; *DNA/metabolism/chemistry/genetics ; Light ; Humans ; },
abstract = {The regulation of CRISPR‒Cas activity is critical for developing advanced biotechnologies. Optical control of CRISPR‒Cas system activity can be achieved by modulation of Cas proteins or guide RNA (gRNA), but these approaches either require complex protein engineering modifications or customization of the optically modulated gRNAs according to the target. Here, we present a method, termed photocleavable phosphorothioate DNA (PC&PS DNA)-mediated regulation of CRISPR‒Cas activity (DNACas), that is versatile and overcomes the limitations of conventional methods. In DNACas, CRISPR‒Cas activity is silenced by the affinity binding of PC&PS DNA and restored through light-triggered chemical bond breakage of PC&PS DNA. The universality of DNACas is demonstrated by adopting the PC&PS DNA to regulate various CRISPR‒Cas enzymes, achieving robust light-switching performance. DNACas is further adopted to develop a light-controlled one-pot LAMP-BrCas12b detection method and a spatiotemporal gene editing strategy. We anticipate that DNACas could be employed to drive various biotechnological advances.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Phosphorothioate Oligonucleotides/metabolism/chemistry/genetics
*DNA/metabolism/chemistry/genetics
Light
Humans

@article {pmid40593661,
year = {2025},
author = {Hwang, HY and Lee, M and Yi, H and Seok, C and Lim, K and Na, YR and Kang, JS and Park, JH and Kim, D},
title = {Engineered Sdd7 cytosine base editors with enhanced specificity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5881},
pmid = {40593661},
issn = {2041-1723},
support = {2020R1A2C2101714//National Research Foundation of Korea (NRF)/ ; RS-2025-00521074//National Research Foundation of Korea (NRF)/ ; HI21C1314, HR22C1363, RS-2024-02507183,21A0202L1-12//Korea Health Industry Development Institute (KHIDI)/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Cytosine Deaminase/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7, a recently developed cytosine deaminase, exhibits high activity across a broad protospacer range but induces unintended off-target effects, including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here, we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this, we engineer two Sdd7 variants, Sdd7e1 and Sdd7e2, enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing, narrowed editing windows, and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity, nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2, especially when delivered via eVLP, as high-fidelity CBEs poised for safe, precise therapeutic genome editing.},
}

*Gene Editing/methods
*Cytosine/metabolism
Humans
CRISPR-Cas Systems/genetics
HEK293 Cells
*Cytosine Deaminase/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40593653,
year = {2025},
author = {Pastuszka, A and Mazzuoli, MV and Crestani, C and Deborde, L and Sismeiro, O and Lemaire, C and Rong, V and Gominet, M and Jacquemet, E and Legendre, R and Lanotte, P and Firon, A},
title = {The virulence regulator CovR boosts CRISPR-Cas9 immunity in Group B Streptococcus.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5678},
pmid = {40593653},
issn = {2041-1723},
support = {ANR-22-CE15-0024//Agence Nationale de la Recherche (French National Research Agency)/ ; LabEx IBEID, ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Streptococcus agalactiae/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Bacterial Proteins/genetics/metabolism/immunology ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Streptococcal Infections/immunology/microbiology ; Promoter Regions, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; Operon ; Host-Pathogen Interactions/immunology/genetics ; Mice ; Animals ; Humans ; },
abstract = {CRISPR-Cas9 immune systems protect bacteria from foreign DNA. However, immune efficiency is constrained by Cas9 off-target cleavages and toxicity. How bacteria regulate Cas9 to maximize protection while preventing autoimmunity is not understood. Here, we show that the master regulator of virulence, CovR, regulates CRISPR-Cas9 immunity against mobile genetic elements in Streptococcus agalactiae, a pathobiont responsible for invasive neonatal infections. We show that CovR binds to and represses a distal promoter of the cas operon, integrating immunity within the virulence regulatory network. The CovR-regulated promoter provides a controlled increase in off-target cleavages to counteract mutations in the target DNA, restores the potency of old immune memory, and stimulates the acquisition of new memory in response to recent infections. Regulation of Cas9 by CovR is conserved at the species level, with lineage specificities suggesting different adaptive trajectories. Altogether, we describe the coordinated regulation of immunity and virulence that enhances the bacterial immune repertoire during host-pathogen interaction.},
}

*Streptococcus agalactiae/genetics/immunology/pathogenicity
*CRISPR-Cas Systems/genetics/immunology
*Bacterial Proteins/genetics/metabolism/immunology
Virulence/genetics
Gene Expression Regulation, Bacterial
Streptococcal Infections/immunology/microbiology
Promoter Regions, Genetic
*CRISPR-Associated Protein 9/genetics/metabolism
Operon
Host-Pathogen Interactions/immunology/genetics
Mice
Animals
Humans

@article {pmid40593649,
year = {2025},
author = {Thorpe, C and Luo, W and Ji, Q and Eggenberger, AL and Chicowski, AS and Xu, W and Sandhu, R and Lee, K and Whitham, SA and Qi, Y and Wang, K and Jiang, S},
title = {Enhancing biolistic plant transformation and genome editing with a flow guiding barrel.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {5624},
pmid = {40593649},
issn = {2041-1723},
support = {2019-67013-29016//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04714//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOW04308//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; IOS-2132693//NSF | BIO | Division of Biological Infrastructure (DBI)/ ; },
mesh = {*Gene Editing/methods ; *Biolistics/methods/instrumentation ; Zea mays/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Triticum/genetics ; *Transformation, Genetic ; Onions/genetics ; Genome, Plant ; Seedlings/genetics ; Genetic Engineering/methods ; },
abstract = {The biolistic delivery system is an essential tool in plant genetic engineering, capable of delivering DNAs, RNAs, and proteins independent of tissue type, genotype, or species. However, its efficiency and consistency remain longstanding challenges despite decades of widespread use. Here, through advanced simulations, we identify gas and particle flow barriers as the root cause of these limitations. We show that a flow guiding barrel (FGB) achieves a 22-fold enhancement in transient transfection efficiency, a 4.5-fold increase in CRISPR-Cas9 ribonucleoprotein editing efficiency in onion epidermis, and a 17-fold improvement in viral infection efficiency in maize seedlings. Furthermore, stable transformation frequency in maize using B104 immature embryos increases over 10-fold, while in planta CRISPR-Cas12a-mediated genome editing efficiency in wheat meristems doubles in both T0 and T1 generations. This study provides insights into the fundamental mechanisms underlying biolistic inefficiency and demonstrates a practical solution that enables broader and more reliable applications in plant genetic engineering.},
}

*Gene Editing/methods
*Biolistics/methods/instrumentation
Zea mays/genetics
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Triticum/genetics
*Transformation, Genetic
Onions/genetics
Genome, Plant
Seedlings/genetics
Genetic Engineering/methods