@article {pmid40811506,
year = {2025},
author = {Pernomian, L and Parente, JM and McCarthy, CG and Wenceslau, CF},
title = {Orphan Under Pressure: GPR146 as a Mechanotransduction Modulator.},
journal = {Circulation research},
volume = {137},
number = {5},
pages = {625-627},
doi = {10.1161/CIRCRESAHA.125.327056},
pmid = {40811506},
issn = {1524-4571},
}

@article {pmid40810510,
year = {2025},
author = {Chen, DF and Roe, LT and Yuping, L and Borges, AL and Zhang, JY and Babbar, P and Maji, S and Stevens, MGV and Correy, GJ and Diolaiti, ME and Smith, DH and Ashworth, A and Stroud, RM and Kelly, MJS and Bondy-Denomy, J and Fraser, JS},
title = {AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.},
journal = {mBio},
volume = {},
number = {},
pages = {e0169825},
doi = {10.1128/mbio.01698-25},
pmid = {40810510},
issn = {2150-7511},
abstract = {Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems, allowing phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors. While enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system. We present a nuclear magnetic resonance (NMR) structure of an AcrIF11 homolog that reveals chemical shift perturbations consistent with NAD (cofactor) binding. In experiments that model both lytic phage replication and MGE/lysogen stability under high targeting pressure, AcrIF11 is a highly potent CRISPR-Cas inhibitor and more robust to Cas protein-level fluctuations than stoichiometric inhibitors. Furthermore, we demonstrate that AcrIF11 is remarkably specific, predominantly ADP-ribosylating Csy1 when expressed in P. aeruginosa. Given the reversible nature of ADP-ribosylation, we hypothesized that ADPr eraser enzymes (macrodomains) could remove ADPr from Csy1, a potential limitation of PTM-based CRISPR inhibition. We demonstrate that a human macrodomain can indeed remove the modification from Csy1 in P. aeruginosa lysate. Together, these experiments connect the in vitro observations of AcrIF11's enzymatic activity to its potent and specific effects in vivo, clarifying the advantages and drawbacks of enzymatic Acrs in the evolutionary arms race between phages and bacteria.IMPORTANCEBacteria have evolved diverse immune systems to prevent phage infection, and, consequently, phages have evolved diverse methods of evading bacterial immune systems. To evade the bacterial CRISPR-Cas immune system, phages encode anti-CRISPR proteins (Acrs). Acrs disable CRISPR-Cas by either stably binding to the CRISPR-Cas complex or by enzymatic modification. However, Acr enzymes have not been characterized in vivo during lytic infection or lysogenic maintenance. Here, we report the benefits and drawbacks of enzymatic inhibition with AcrIF11, an ADP-ribosyltransferase. Under "high pressure" scenarios such as high CRISPR targeting or CRISPR-Cas overexpression, AcrIF11 outperforms a strong, stable binding Acr by very specifically modifying the Cas8 protein, but nothing else in the cell. AcrIF11 additionally stabilizes lysogeny effectively, but the ADP-ribose modification can potentially be removed by macrodomains, which are ADP-ribose eraser enzymes. AcrIF11 is therefore a potent and widespread plasmid/phage-encoded inhibitor of Type I-F CRISPR-Cas systems with catalytic activity.},
}

@article {pmid40808298,
year = {2025},
author = {Andersen, S and Wolff, JH and Skov, TW and Janns, JH and Davis, LJ and Haldrup, JH and Haslund, D and Revenfeld, AL and Relkovic, D and Møller, BK and Lund, J and Bak, RO and Thomsen, EA and Mikkelsen, JG},
title = {Gene editing in hematopoietic stem cells by co-delivery of Cas9/sgRNA ribonucleoprotein and templates for homology-directed repair in 'all-in-one' lentivirus-derived nanoparticles.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
doi = {10.1093/nar/gkaf767},
pmid = {40808298},
issn = {1362-4962},
support = {//Innovation Fund Denmark/ ; //Novo Nordisk A/S/ ; 8056-00010A//Innovation Fund Denmark/ ; NNF22OC0080684//Novo Nordisk Foundation/ ; 4285-00102B//Independent Research Fund Denmark/ ; CF21-0363//Carlsberg Foundation/ ; },
abstract = {Repair of double-strand DNA breaks generated by site-directed endonucleases, like Cas9, is the hallmark of gene editing based on homology-directed repair (HDR). HDR uses an exogenous DNA template to restore the cleaved DNA sequence and can facilitate specific gene corrections as well as insertion of genes or partial complementary DNA (cDNA) sequences. For CRISPR/Cas-directed gene editing, co-administration of the Cas9/single guide RNA (sgRNA) ribonucleoprotein (RNP) complex and a DNA template typically involves two different delivery strategies or different types of vehicles. This requires exquisite timing of delivery and may potentially challenge safety and therapeutic applicability. There is a need therefore for technologies that can ferry complete editing tool kits into cells. Here, we demonstrate the use of lentivirus-derived nanoparticles (LVNPs) to transport both RNP complexes and vector RNA, which upon reverse transcription serves as a repair template for HDR-directed gene editing. Such 'all-in-one' LVNPs support targeted gene insertion with reduced off-target effects relative to nucleofection procedures. We show potent editing in the HBB gene in human erythroid progenitor cells as well as HDR-directed editing in hematopoietic stem and progenitor cells. Our findings mark a first step toward using a single virus-derived vehicle for delivering a full HDR gene editing kit.},
}

@article {pmid40807307,
year = {2025},
author = {Alessa, O and Aiba, Y and Arbaah, M and Hidaka, Y and Watanabe, S and Miyanaga, K and Wannigama, DL and Cui, L},
title = {Synthetic and Functional Engineering of Bacteriophages: Approaches for Tailored Bactericidal, Diagnostic, and Delivery Platforms.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {15},
pages = {},
doi = {10.3390/molecules30153132},
pmid = {40807307},
issn = {1420-3049},
support = {(grant No. JP25ae0121045, JP25gm1610002, and JP25fk0108698), (Grant No. JP24H00662, JP24K22027, JP25K13511, and JP25K19273) and (JPJ009237).//This research was funded by the Japan Agency for Medical Research and Development JSPS KAKENHI and partially by Moonshot R&D Program for Agriculture, Forestry, Fisheries/ ; },
abstract = {Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription-translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology.},
}

@article {pmid40806631,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Choi, K and Daurov, D and Daurova, A and Zhambakin, K and Shamekova, M},
title = {CRISPR-Cas Gene Editing Technology in Potato.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157496},
pmid = {40806631},
issn = {1422-0067},
support = {BR21882269//The Science Committee of the Ministry of Science and Higher Education, Republic of Kazakhstan/ ; },
abstract = {Potato (Solanum tuberosum L.) is one of the most important food crops in the world, ranking fourth after rice, maize, and wheat. Potatoes are exposed to biotic and abiotic environmental factors, which lead to economic losses and increase the possibility of food security threats in many countries. Traditional potato breeding faces several challenges, primarily due to its genetic complexity and the time-consuming nature of the process. Therefore, gene editing-CRISPR-Cas technology-allows for more precise and rapid changes to the potato genome, which can speed up the breeding process and lead to more effective varieties. In this review, we consider CRISPR-Cas technology as a potential tool for plant breeding strategies to ensure global food security. This review summarizes in detail current and potential technological breakthroughs that open new opportunities for the use of CRISPR-Cas technology for potato breeding, as well as for increasing resistance to abiotic and biotic stresses, and improving potato tuber quality. In addition, the review discusses the challenges and future perspectives of the CRISPR-Cas system in the prospects of the development of potato production and the regulation of gene-edited crops in different countries around the world.},
}

@article {pmid40806610,
year = {2025},
author = {Shaposhnikov, LA and Rozanov, AS and Sazonov, AE},
title = {Genome-Editing Tools for Lactic Acid Bacteria: Past Achievements, Current Platforms, and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157483},
pmid = {40806610},
issn = {1422-0067},
support = {№18-03//Sirius University of Science and Technology/ ; },
abstract = {Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications.},
}

@article {pmid40806599,
year = {2025},
author = {Yang, L and Lu, Q},
title = {Beyond Cutting: CRISPR-Driven Synthetic Biology Toolkit for Next-Generation Microalgal Metabolic Engineering.},
journal = {International journal of molecular sciences},
volume = {26},
number = {15},
pages = {},
doi = {10.3390/ijms26157470},
pmid = {40806599},
issn = {1422-0067},
abstract = {Microalgae, with their unparalleled capabilities for sunlight-driven growth, CO2 fixation, and synthesis of diverse high-value compounds, represent sustainable cell factories for a circular bioeconomy. However, industrial deployment has been hindered by biological constraints and the inadequacy of conventional genetic tools. The advent of CRISPR-Cas systems initially provided precise gene editing via targeted DNA cleavage. This review argues that the true transformative potential lies in moving decisively beyond cutting to harness CRISPR as a versatile synthetic biology "Swiss Army Knife". We synthesize the rapid evolution of CRISPR-derived tools-including transcriptional modulators (CRISPRa/i), epigenome editors, base/prime editors, multiplexed systems, and biosensor-integrated logic gates-and their revolutionary applications in microalgal engineering. These tools enable tunable gene expression, stable epigenetic reprogramming, DSB-free nucleotide-level precision editing, coordinated rewiring of complex metabolic networks, and dynamic, autonomous control in response to environmental cues. We critically evaluate their deployment to enhance photosynthesis, boost lipid/biofuel production, engineer high-value compound pathways (carotenoids, PUFAs, proteins), improve stress resilience, and optimize carbon utilization. Persistent challenges-species-specific tool optimization, delivery efficiency, genetic stability, scalability, and biosafety-are analyzed, alongside emerging solutions and future directions integrating AI, automation, and multi-omics. The strategic integration of this CRISPR toolkit unlocks the potential to engineer robust, high-productivity microalgal cell factories, finally realizing their promise as sustainable platforms for next-generation biomanufacturing.},
}

@article {pmid40804951,
year = {2025},
author = {Li, G and Zhou, X and Zhu, G and Pan, Y and Yan, J and Meng, J and Ye, T and Cheng, Y and Liu, C and Gu, Z},
title = {Macrobrachium rosenbergii Genome Editing Breeding with CRISPR-Cas Nucleases, Base Editors, and Prime Editors.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {15},
pages = {},
doi = {10.3390/ani15152161},
pmid = {40804951},
issn = {2076-2615},
support = {2024SSYS0101//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 32401241//National Natural Science Foundation of China/ ; },
abstract = {This review focuses on CRISPR genome editing technology, particularly its application in the study of Macrobrachium rosenbergii (M. rosenbergii). It first elaborates on the basic principles and mechanisms of CRISPR-Cas9 technology, base editors, and prime editors. Then, it explores the application of this technology in M. rosenbergii breeding, including improving growth rate, enhancing disease resistance, and sex control. Additionally, it introduces the progress of genome editing technology in M. rosenbergii, epidemiology and pathogenesis, diagnostic techniques, analyzes the opportunities and challenges it faces, reviews the historical evolution, and looks ahead to future development directions. CRISPR technology has brought new opportunities to the research and industrial development of M. rosenbergii, but it also needs to address numerous technical and safety challenges.},
}

@article {pmid40804123,
year = {2025},
author = {Yu, M and Ai, L and Wang, B and Lian, S and Ip, L and Liu, J and Li, L and Tsai, SQ and Kleinstiver, BP and Zheng, Z},
title = {GenomePAM directs PAM characterization and engineering of CRISPR-Cas nucleases using mammalian genome repeats.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40804123},
issn = {2157-846X},
support = {202001418//Vetenskapsrådet (Swedish Research Council)/ ; 11103024//Research Grants Council, University Grants Committee (RGC, UGC)/ ; T12-101/23-N//Research Grants Council, University Grants Committee (RGC, UGC)/ ; DP2-CA281401//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
abstract = {Characterizing the protospacer adjacent motif (PAM) requirements of different Cas enzymes is a bottleneck in the discovery of Cas proteins and their engineered variants in mammalian cell contexts. Here, to overcome this challenge and to enable more scalable characterization of PAM preferences, we develop a method named GenomePAM that allows for direct PAM characterization in mammalian cells. GenomePAM leverages genomic repetitive sequences as target sites and does not require protein purification or synthetic oligos. GenomePAM uses a 20-nt protospacer that occurs ~16,942 times in every human diploid cell and is flanked by nearly random sequences. We demonstrate that GenomePAM can accurately characterize the PAM requirement of type II and type V nucleases, including the minimal PAM requirement of the near-PAMless SpRY and extended PAM for CjCas9. Beyond PAM characterization, GenomePAM allows for simultaneous comparison of activities and fidelities among different Cas nucleases on thousands of match and mismatch sites across the genome using a single gRNA and provides insight into the genome-wide chromatin accessibility profiles in different cell types.},
}

@article {pmid40809473,
year = {2023},
author = {Mecacci, S and Torregrosa-Barragán, L and Asin-Garcia, E and Smith, RW},
title = {Multilayered safety framework for living diagnostics in the colon.},
journal = {Frontiers in systems biology},
volume = {3},
number = {},
pages = {1240040},
pmid = {40809473},
issn = {2674-0702},
abstract = {Introduction: Colorectal cancer is the second most deadly cancer worldwide. Current screening methods have low detection rates and frequently provide false positive results, leading to missed diagnoses or unnecessary colonoscopies. To tackle this issue, the Wageningen UR iGEM team from 2022 developed "Colourectal", a living diagnostic tool for colorectal cancer. Following a synthetic biology approach, the project used an engineered Escherichia coli Nissle 1917 strain capable of binding to tumour cells that detects two distinct cancer biomarkers, and secretes a coloured protein observable in stool. Due to the utilization of genetically modified bacteria in vivo, precautionary biosafety measures were included within a three level safe-by-design strategy. Results: The first genetic safeguard ensured confinement of the living diagnostic to the colon environment by implementing auxotrophy to mucin that is abundant in the colon lining. For this, a synthetic chimeric receptor was generated to ensure expression of essential genes in the presence of mucin. The second strategy limited the viability of the engineered bacteria to the human body, preventing proliferation in open environments. The use of a temperature sensitive kill switch induced bacterial cell death at temperatures below 37°C. The third biocontainment strategy was installed as an emergency kill switch to stop the Colourectal test at any point. By inducing a highly genotoxic response through CRISPR-Cas-mediated DNA degradation, cell death of E. coli Nissle is triggered. Discussion: While the use of engineered microorganisms in human applications is not yet a reality, the safety considerations of our multi-layered strategy provide a framework for the development of future living diagnostic tools.},
}

@article {pmid40803977,
year = {2025},
author = {Li, L and Zhang, Z and Zhang, B},
title = {CRISPR meets AlphaFold: guiding SWEET10-enhanced oil production.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2025.08.001},
pmid = {40803977},
issn = {1878-4372},
abstract = {Enhancing seed oil content significantly benefits both human welfare and environmental sustainability. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) and artificial intelligence (AI) are transformative tools for crop trait improvement. A recent study by Wang and colleagues reported that AlphaFold-guided CRISPR genome editing of SWEET10 boosts oil contents, highlighting a breakthrough in precision crop engineering.},
}

@article {pmid40803657,
year = {2025},
author = {Ceasar, SA and Ebeed, HT and Ramakrishnan, M and García-Caparrós, P and Ignacimuthu, S},
title = {Understanding low-phosphate stress responses in plants: Opportunities for genome editing to improve phosphorous use efficiency (PUE).},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108686},
doi = {10.1016/j.biotechadv.2025.108686},
pmid = {40803657},
issn = {1873-1899},
abstract = {Phosphorus (P) is a critical macronutrient essential for plant growth, yet its availability in soil is often limited due to poor mobility and fixation with metal ions due to acidic or alkaline soils. Plants have evolved complex adaptive responses to overcome phosphate (Pi) deficiency. Recent advancements in genome editing, particularly CRISPR/Cas tools, offer opportunities to enhance these adaptive traits for sustainable agriculture. This review consolidates current understanding of low Pi stress signaling pathways, including morphological (root architecture changes), biochemical (hormone regulation, lipid modification, organic acid exudation), and molecular (transcription factors (TFs), phosphate transporters, and microRNAs), and identifies prime candidate genes for genome editing applications. Key regulators such as phosphate transporter (PHT, PHO1), TFs (PHR1, WRKYs) and microRNAs (miR399/827) manage Pi uptake, redistribution, and signaling. Genome editing strategies targeting root-specific traits, hormonal integration, lipid remodeling, and transcriptional regulation are discussed as viable ways for improving phosphorous use efficiency (PUE). Harnessing CRISPR/Cas tools can lead to the development of crops with optimized PUE, reduced dependency on synthetic fertilizers, and improved adaptability to Pi-deficient soils. The review provides a comprehensive roadmap for researchers and breeders to apply CRISPR/Cas technology toward building next-generation crops capable of thriving under low Pi conditions.},
}

@article {pmid40803478,
year = {2025},
author = {Jian, A and Zhao, G and Wang, Y and Wang, S and Li, N},
title = {Watershed Year of Cell and Gene Therapy (CGT): A Review of 2024 CGT Approvals.},
journal = {Cancer letters},
volume = {},
number = {},
pages = {217980},
doi = {10.1016/j.canlet.2025.217980},
pmid = {40803478},
issn = {1872-7980},
abstract = {The year 2024 is a pivotal year for therapeutic breakthroughs in human diseases, alongside with an uprising growth in precision medicine, especially in cell and gene therapies (CGTs), marked by an unprecedented approval number of 13 novel CGTs authorized by the U.S. FDA, China's NMPA, E.U. EMA, and Japan's PMDA. 2024 is also a year of many firsts: the first CRISPR therapy, the first MRI-guided intracranial AAV delivery gene therapy, and the first tumor infiltration lymphocyte therapy, opening a whole new chapter of clinical translation of innovations in gene-editing and cell technologies. CGTs represent an emerging translational modality in precision medicine through utilization of cellular or genetic materials to treat or prevent disease, offering curative potential for previously refractory diseases. Despite thriving in CGTs' development, comprehensive analyses of 2024 approvals remain absent. This review employs a quadruple axle comparative framework to analyze 2024-approved CGTs in China and USA, integrating mechanistic innovation with gradually improving regulatory advancements. Raw data was extracted from official agency databases, pivotal trials, and manufacturing reports. This work further delineates how mechanistic diversity converges with regulatory agility to redefine therapeutic development.},
}

@article {pmid40803134,
year = {2025},
author = {Wu, F and Campbell, BC and Greenfield, P and Hose, GC and Midgley, DJ and George, SC},
title = {There and back again: Genomic insights into microbial life in a recirculating petroleum refinery wastewater biotreatment system.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128299},
doi = {10.1016/j.micres.2025.128299},
pmid = {40803134},
issn = {1618-0623},
abstract = {Petroleum refinery wastewater biotreatment relies on microbes to remediate carbon, nitrogen, and sulfur compounds, yet their life strategies and ecological roles remain unclear. This study characterises the ecological functions of 20 metagenome-assembled genomes (MAGs) from a full-scale petroleum refinery wastewater treatment plant in southern China. The taxonomic identity, nutrient metabolism genes (including C/N/S cycling), carbohydrate-active enzymes, and CRISPR-Cas systems of these MAGs were analysed. The recovered MAGs represented bacteria primarily from the Pseudomonadota and Bacteroidota phyla. The major carbon sources for the represented organisms are likely aromatic and aliphatic compounds, as well as carbohydrates including peptidoglycan, chitin, and starch. Almost all MAGs contained genes for nitrate or nitrite reduction, while metabolic pathways for sulfur metabolism were generally less prevalent. Meiothermus sp. bin.89 was the most metabolically versatile MAG. This organism possessed genes that allowed it to recycle biomass, break down aliphatic and monoaromatic compounds, and perform anaerobic respiration using nitrate. However, it was likely the most susceptible to viral predation, as indicated by the high abundance of CRISPR spacers. Overall, the results revealed that stress-tolerant ecological traits were common among organisms in this microbiome, showcasing the ability of the microbes to obtain carbon from aromatic and aliphatic compounds. This study provides a substantial contribution towards future efforts in optimising microbiome stability for pollutant removal in petroleum refinery wastewater biotreatment systems.},
}

@article {pmid40802883,
year = {2025},
author = {Song, N and Wang, L and Zhang, L and Tian, G and Yao, C and Yang, D},
title = {Precision Delivery of CRISPR/Cas Systems via DNA Nanostructures for Gene Therapy and Intracellular Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {e202500357},
doi = {10.1002/cbic.202500357},
pmid = {40802883},
issn = {1439-7633},
support = {22225505//National Natural Science Foundation of China/ ; 22322407//National Natural Science Foundation of China/ ; 22174097//National Natural Science Foundation of China/ ; 224B2505//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR/Cas system represents a transformative breakthrough in genome editing technology, featuring three principal effector proteins with distinct functionalities: Cas9, which induces site-specific double-strand breaks guided by a single guide RNA, enabling precise gene knockout and knock-in modifications; Cas12, which mediates targeted DNA cleavage through cis-activity while exhibiting nonspecific trans-cleavage of single-stranded DNA, a property exploited for ultrasensitive nucleic acid detection in molecular diagnostics; and Cas13, an RNA-guided RNase that specifically degrades complementary RNA transcripts, demonstrating significant potential for antiviral therapies and transcriptome regulation. Despite these advances, the clinical translation of CRISPR/Cas systems faces substantial challenges, particularly in achieving efficient and controllable delivery. This reviewsystematically examines current delivery modalities for CRISPR/Cas systems, with particular emphasis on the implementation of DNA-based functional materials as advanced delivery vehicles. The integration of multifunctional DNA nanostructures with diverse CRISPR/Cas systems may facilitate the development of integrated theranostic platforms, thereby advancing precision medicine through synergistic bioengineering approaches.},
}

@article {pmid40801580,
year = {2025},
author = {Leal, AF and Prieto, LE and Pachajoa, H},
title = {CRISPR/Cas-Based Ex Vivo Gene Therapy and Lysosomal Storage Disorders: A Perspective Beyond Cas9.},
journal = {Cells},
volume = {14},
number = {15},
pages = {},
doi = {10.3390/cells14151147},
pmid = {40801580},
issn = {2073-4409},
abstract = {Lysosomal storage disorders (LSDs) are inherited metabolic conditions characterized by lysosomal enzyme deficiencies leading to substrate accumulation. As genetic diseases, LSDs can be treated with gene therapies (GT), including the CRISPR/Cas systems. The CRISPR/Cas systems enable precise and programmable genome editing, leading to targeted modifications at specific genomic loci. While the classical CRISPR/Cas9 system has been extensively used to generate LSD disease models and correct disease-associated genetic alterations through homologous recombination (HR), recently described Cas proteins as well as CRISPR/Cas9-derived strategies such as base editing, prime editing, and homology-independent targeted integration (HITI) offer a novel way to develop innovative treatments for LSDs. The direct administration of the CRISPR/Cas9 system remains the primary strategy evaluated in several LSDs; nevertheless, the ex vivo CRISPR/Cas9-based approach has been recently explored, primarily in central nervous system-affecting LSDs. Ex vivo approaches involve genetically modifying, in theory, any patient cells in the laboratory and reintroducing them into the patient to provide a therapeutic effect. This manuscript reviews the molecular aspects of the CRISPR/Cas technology and its implementation in ex vivo strategies for LSDs while discussing novel approaches beyond the classical CRISPR/Cas9 system.},
}

@article {pmid40801057,
year = {2025},
author = {Talibli, F and Voß, B},
title = {Metagenomic CRISPR Array Analysis Tool: a novel graph-based approach to finding CRISPR arrays in metagenomic datasets.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf016},
pmid = {40801057},
issn = {2633-6693},
abstract = {Clustered Regularly Interspersed Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas) is a bacterial immune system also famous for its use in genome editing. The diversity of known systems could be significantly increased by metagenomic data. Here we present the Metagenomic CRISPR Array Analysis Tool (MCAAT), a highly sensitive algorithm for finding CRISPR arrays in unassembled metagenomic data. It takes advantage of the properties of CRISPR arrays that form multicycles in de Bruijn graphs. We show that MCAAT reliably predicts CRISPR arrays in bacterial genome sequences and that its assembly-free graph-based strategy outperforms assembly-based workflows and other assembly-free methods on synthetic and real metagenomes. Our new approach will help to increase the diversity of known CRISPR-Cas systems and enable studies of spacer evolution within metagenomic data sets.},
}

@article {pmid40796771,
year = {2025},
author = {Wolff, JH and Skov, TW and Haslund, D and Dorset, SR and Revenfeld, ALS and Aussel, C and Jørgensen, SE and Holm, M and Thomsen, MK and Ammann, S and Cathomen, T and Mogensen, TH and Møller, BK and Bak, RO and Mikkelsen, JG},
title = {Targeted gene editing and near-universal cDNA insertion of CYBA and CYBB as a treatment for chronic granulomatous disease.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7475},
pmid = {40796771},
issn = {2041-1723},
support = {8056-00010A//Innovationsfonden (Innovation Fund Denmark)/ ; NNF220C0080684//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; CF21-0363//Carlsbergfondet (Carlsberg Foundation)/ ; CA 311/4-1 & FANEDIT/EJPRD20-209//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Chronic granulomatous disease (CGD) is a severe inborn error of immunity caused by NADPH oxidase defects. Here, we develop CRISPR/Cas9-based gene editing strategies for correction of variants in the CYBA and CYBB genes causing CGD. For X-linked CGD, we also develop a near-universal gene editing strategy by targeted integration of a truncated CYBB cDNA in CD34[+] hematopoietic stem and progenitor cells (HSPCs). Throughout, off-target editing and chromosomal translocations are evident, which negatively impact the ability of gene-edited HSPCs to engraft in immunodeficient mice. However, by employing a high-fidelity Cas9 to minimize off-target editing, we demonstrate restoration of the multilineage engraftment potential of gene-edited HSPCs. Moreover, to further improve safety, we develop a D10A Cas9n editing approach with no detectable off-target activity or chromosomal translocations. Collectively, through risk assessments of different gene editing approaches, we present a D10A Cas9n-based strategy with improved safety, offering a potentially curative treatment for CGD patients.},
}

@article {pmid40796304,
year = {2025},
author = {Qing, Y and Liao, Z and An, D and Zeng, Y and Zhu, Q and Zhang, X},
title = {Comparative genomics reveals the genetic diversity and plasticity of Clostridium tertium.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxaf201},
pmid = {40796304},
issn = {1365-2672},
abstract = {AIMS: Clostridium tertium, increasingly recognized as the emerging human pathogen frequently isolated from environmental and clinical specimens, remains genetically underexplored despite its clinical relevance. This study aims to explore the genetic characteristics of C. tertium by genomic analysis.

METHODS AND RESULTS: This study presented a comprehensive genomic investigation of 45 C. tertium strains from the GenBank database. Genome sizes (3.27-4.55 Mbp) and coding gene counts varied markedly across strains. Phylogenetic analyses based on 16S rRNA gene and core genome uncovered distinct intra-species lineages, including evolutionarily divergent clusters likely shaped by niche specialization. Pan-genomic analysis confirmed an open genome, with accessory and strain-specific genes enriched in functions related to environmental adaptation and regulation. Functional annotation further identified diverse virulence factor genes (e.g. clpP, nagK) and antibiotic resistance genes (e.g. vatB, tetA(P)) co-occurring with mobile genetic elements (MGEs), suggesting that horizontal gene transfer (HGT) may be a key driver of genome plasticity in C. tertium. Notably, one-third of the strains carried CRISPR-Cas systems, indicating the defense potential against exogenous genetic elements.

CONCLUSIONS: C. tertium exhibited extensive genetic diversity and genome plasticity, probably driven by MGE-mediated HGT, defense mechanisms of CRISPR-Cas systems, and functional adaptation related to virulence and resistance. These traits may underlie its ability to colonize diverse environments and acquire pathogenicity and resistance.},
}

@article {pmid40794864,
year = {2025},
author = {Wang, F and Zhao, P and Bi, X and Zheng, R and Tian, X and Xu, J and Jiang, S and Li, G and Shen, Y and She, Q},
title = {Cyclic tetraadenylate binding induces dimerization of protein dimers to activate a CRISPR-associated PIN nuclease.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf744},
pmid = {40794864},
issn = {1362-4962},
support = {2020YFA0906800//National Key R&D Program of China/ ; 31771380//National Natural Science Foundation of China/ ; 32270040//National Natural Science Foundation of China/ ; 32393972//National Natural Science Foundation of China/ ; SKLMTFCP-2023-05//Frontiers and Challenges/ ; ZR2024QC307//State Key Laboratory of Microbial Technology/ ; //Shandong Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; Protein Multimerization ; *Archaeal Proteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Protein Binding ; *Adenine Nucleotides/metabolism/chemistry ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Crystallography, X-Ray ; Protein Domains ; },
abstract = {Type III CRISPR-Cas systems synthesize cyclic oligoadenylates (cOAs), the second messengers that bind to the CARF (CRISPR-associated Rossman fold) sensor domain and allosterically activate the effector domain of CRISPR ancillary effectors to mediate antiviral defense. An arsenal of such effectors has been identified, but only a minority of them have been characterized thus far. Here, CaPN (a CRISPR-associated PIN domain nuclease), a novel effector protein encoded by Saccharolobus islandicus, was characterized. Biochemical characterization of CaPN revealed that the CARF domain senses cA4 (cyclic tetraadenylate), and its binding to the CARF domain activates the PIN domain for robust RNA cleavage. Genetic assay showed that CaPN mediates growth arrest/cell death to its archaeal host upon cA4 sensing. Determination of the crystal structures of CaPN in apo and in the cA4-bound form revealed that cA4-CARF interactions trigger the conformational changes, leading to the dimerization of the CaPN dimers. These structural changes reposition D296, one of the active site residues in the catalytic pocket, to yield an active PIN domain nuclease. Together, these results unveil a novel molecular mechanism for the activation of cOA-activated Cas ancillary RNases in the CRISPR signaling pathway.},
}

*CRISPR-Cas Systems
Protein Multimerization
*Archaeal Proteins/chemistry/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Protein Binding
*Adenine Nucleotides/metabolism/chemistry
Models, Molecular
*Endonucleases/metabolism/chemistry/genetics
Crystallography, X-Ray
Protein Domains

@article {pmid40794863,
year = {2025},
author = {Brenker, L and Aschenbrenner, S and Bubeck, F and Staykov, K and Gebhardt, C and Wolf, B and Jendrusch, M and Kröll, AS and Becker, J and Ambiel, I and Fackler, OT and Grimm, D and Mathony, J and Niopek, D},
title = {A versatile anti-CRISPR platform for opto- and chemogenetic control of CRISPR-Cas9 and Cas12 across a wide range of orthologs.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf752},
pmid = {40794863},
issn = {1362-4962},
support = {453202693//German Research Foundation/ ; //Aventis foundation/ ; //Rolf. M. Schwiete Stiftung/ ; //Ministry of Science, Research and Culture Baden-Württemberg/ ; },
mesh = {*CRISPR-Cas Systems/genetics/drug effects ; *Gene Editing/methods ; Humans ; *Optogenetics/methods ; *CRISPR-Associated Proteins/antagonists & inhibitors/genetics/metabolism ; CRISPR-Associated Protein 9/antagonists & inhibitors/genetics ; Ligands ; Bacterial Proteins/genetics/metabolism ; HEK293 Cells ; Chemogenetics ; },
abstract = {CRISPR-Cas technologies have revolutionized life sciences by enabling programmable genome editing across diverse organisms. Achieving dynamic and precise control over CRISPR-Cas activity with exogenous triggers, such as light or chemical ligands, remains an important need. Existing tools for CRISPR-Cas control are often limited to specific Cas orthologs or selected applications, restricting their versatility. Anti-CRISPR (Acr) proteins are natural inhibitors of CRISPR-Cas systems and provide a flexible regulatory layer but are constitutively active in their native forms. In this study, we built on our previously reported concept for optogenetic CRISPR-Cas control with engineered, light-switchable anti-CRISPR proteins and expanded it from ortholog-specific Acrs towards AcrIIA5 and AcrVA1, broad-spectrum inhibitors of CRISPR-Cas9 and CRISPR-Cas12a, respectively. We then conceived and implemented a novel, chemogenetic anti-CRISPR platform based on engineered, circularly permuted ligand receptor domains, that together respond to six clinically relevant drugs. The resulting toolbox achieves both optogenetic and chemogenetic control of genome editing in human cells with a wide range of CRISPR-Cas effectors, including type II-A and II-C CRISPR-Cas9s, and CRISPR-Cas12a. In sum, this work establishes a versatile platform for the multidimensional control of CRISPR-Cas systems, with immediate applications in basic research and biotechnology, and with the potential for therapeutic use in the future.},
}

*CRISPR-Cas Systems/genetics/drug effects
*Gene Editing/methods
Humans
*Optogenetics/methods
*CRISPR-Associated Proteins/antagonists & inhibitors/genetics/metabolism
CRISPR-Associated Protein 9/antagonists & inhibitors/genetics
Ligands
Bacterial Proteins/genetics/metabolism
HEK293 Cells
Chemogenetics

@article {pmid40794700,
year = {2025},
author = {Tagliaferri, TL and Krüttgen, A and Mendes, TAO and Gonçalves Dos Santos, S and Horz, HP},
title = {CRISPR-Cas9 targeting the blaKPC gene in a clinical isolate of Klebsiella michiganensis: Reduction of imipenem resistance and changes in genomic carbapenem resistance determinants.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0328521},
doi = {10.1371/journal.pone.0328521},
pmid = {40794700},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; *Klebsiella/genetics/drug effects/isolation & purification ; *Bacterial Proteins/genetics ; *Imipenem/pharmacology ; *beta-Lactamases/genetics ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Escherichia coli/genetics/drug effects ; Plasmids/genetics ; *Drug Resistance, Bacterial/genetics ; Porins/genetics ; Klebsiella Infections/microbiology/drug therapy ; Carbapenems/pharmacology ; },
abstract = {The CRISPR-Cas technology can be used to disable drug resistance genes. Since carbapenem resistance can be mediated by multiple resistance determinants, we here investigated the extent of re-sensizitation when targeting the blaKPC carbapenemase gene and assessed possible effects on porins and efflux pumps. While full re-sensitization was achieved in a laboratory strain of Escherichia coli solely equipped with blaKPC, resistance reduction in a clinical isolate of Klebsiella michiganensis was achieved in 63% of analyzed transformants, which was a consequence of plasmid copy number reduction and decreased blaKPC gene expression. Damages in the Cas9, as well as alterations in carbapenem-resistance promoting genes including ompK36 downregulation and mutations in the acrB gene were found, likely preventing more efficient re-sensitization. Hence, interference with a single resistance gene promoted the emergence of clonal variants that exhibit alterations in outer membrane proteins. Those bacterial countermeasures, however, were not sufficient to restore the original carbapenem-resistant phenotype.},
}

*CRISPR-Cas Systems/genetics
*Klebsiella/genetics/drug effects/isolation & purification
*Bacterial Proteins/genetics
*Imipenem/pharmacology
*beta-Lactamases/genetics
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Humans
Escherichia coli/genetics/drug effects
Plasmids/genetics
*Drug Resistance, Bacterial/genetics
Porins/genetics
Klebsiella Infections/microbiology/drug therapy
Carbapenems/pharmacology

@article {pmid40794116,
year = {2025},
author = {Cai, R and Chai, N and Zhang, J and Tan, J and Liu, YG and Zhu, Q and Zeng, D},
title = {CRISPR/Cas system-mediated transgene-free or DNA-free genome editing in plants.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {9},
pages = {210},
pmid = {40794116},
issn = {1432-2242},
support = {32272120//National Natural Science Foundation of China/ ; 2022YFF1002800//National Key Research and Development Program of China/ ; 2024GXNSFBA010386//Guangxi Natural Science Foundation Youth Science Fund Project/ ; 2024KY0071//The Project for Enhancing Young and Middle-aged Teacher's Research Basis Ability in Colleges of Guangxi/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; *Crops, Agricultural/genetics ; Plant Breeding ; },
abstract = {CRISPR/Cas-based genome-editing technology serves as a powerful and versatile tool for genome modification. It has been broadly utilized in crop breeding to enhance traits such as yield, various quality attributes, and biotic and abiotic stress tolerance. Because of public safety concerns over genetically modified organisms (GMOs), many countries have established stringent regulatory policies for genetically modified plants, dramatically limiting the application of related products. However, genome editing in stably transformed plants can result in transgene-free progeny through self-pollination or hybridization or yield DNA-free gene-edited plants via transient transformation. These edited plants materially differ from GMOs and are referred to as genome-edited organisms (GEOs). GEOs have the potential to alleviate regulatory burdens and aid in commercialization. Various methods have been developed to expedite the creation of transgene-free or DNA-free GEOs. This review summarizes the various strategies for creating these types of GEOs based on the CRISPR/Cas systems. It also covers the advantages and drawbacks of these strategies. Additionally, we examine off-target effects and mitigation strategies for plant genome editing and outline regulatory policies for gene-edited crops in selected countries and regions. We hope this review offers valuable references for the advancement of transgene-free and DNA-free GEOs.},
}

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

@article {pmid40793766,
year = {2025},
author = {Xu, H-W and Wang, X-Y and Wei, Y and Cao, Y and Wang, S-G and Xia, P-F},
title = {Pathway crosstalk enables degradation of aromatic compounds in marine Roseobacter clade bacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0097825},
doi = {10.1128/aem.00978-25},
pmid = {40793766},
issn = {1098-5336},
abstract = {Aromatic compounds are essential raw materials for almost all sectors of human societies but also persistent environmental pollutants recalcitrant to biodegradation. The ocean serves as a significant sink for these compounds, while their biological conversion routes remain poorly understood, hindering a comprehensive understanding of the marine carbon cycle and advancements in bioremediation and biological carbon upcycling. Here, we report the degradation pathway of aromatic molecules in the marine Roseobacter clade bacteria through multi-omics analysis and CRISPR-Cas-based genome editing. Using Roseovarius nubinhibens and 4-hydroxybenzoate (4HB) as representatives, we identified the transport of 4HB via TRAP, ABC, and MFS transporters. Then, we deciphered the integral β-ketoadipate pathway responsible for aromatic degradation. Next, we discovered a distinct pathway crosstalk at the final thiolation step, which serves as an intersection node of different pathways catalyzed by the 3-oxoadipyl-CoA thiolase from the β-ketoadipate pathway and the acetyl-CoA C-acetyltransferase and acetyl-CoA C-acyltransferase from the β-oxidation pathway. Finally, we proposed R. nubinhibens as a novel marine platform for systems-level interrogation and bioprospecting. Our study provides a foundation for leveraging Roseobacter clade bacteria as novel chassis for environmental and industrial innovations.IMPORTANCEAromatic compounds lie in an essential node of carbon cycling in both natural and engineered systems. Marine bacteria orchestrate the cycling of aromatic compounds in the ocean and, as emerging chassis, have shown unusual potentials in the degradation and valorization of aromatics. However, the corresponding metabolic pathway in marine bacteria remains poorly interpreted over decades, hindering further scientific interrogation and engineering practices. Here, we deciphered the complete degradation pathway of aromatic compounds in the marine Roseobacter clade bacteria and established a marine platform for systems and synthetic biology. Our study provides a paradigm for biological interrogation with combined multi-omics and the cutting-edge CRISPR-Cas approaches, laying a foundation for biological innovations with marine bacteria.},
}

@article {pmid40792102,
year = {2025},
author = {Chen, X and Xu, L and Luo, Z and Wang, L and Wang, Z and Li, Y and Jiao, X and Li, Q},
title = {Prevalence and genomic insights into type III-A CRISPR-Cas system acquisition in global Staphylococcus argenteus strains.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1644286},
pmid = {40792102},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems/genetics ; *Staphylococcus/genetics/classification/isolation & purification ; Humans ; *Genome, Bacterial ; *Staphylococcal Infections/microbiology/epidemiology/veterinary ; Animals ; Genomics ; DNA Transposable Elements ; Prevalence ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {INTRODUCTION: The CRISPR-Cas system serves as a defense mechanism in bacteria and archaea, protecting them against the invasion of mobile genetic elements. Staphylococcus argenteus, a Gram-positive bacterium that diverged from Staphylococcus aureus, is characterized by the rare presence of the CRISPR-Cas system in only a few isolates.

METHODS: In this study, we analyzed the prevalence of the type III-A CRISPR-Cas system in 368 S. argenteus genome sequences from animals, food sources, and humans across 26 countries, available in public database.

RESULTS: Our findings revealed that 44.0% of these strains carry this immune system, with 98.1% of them belonging to the sequence type 2250 (ST2250). Genomic localization analysis indicated that the CRISPR-Cas is closely associated with SCCmec (mecA-ΔmecR1-IS1272-ccrB2-ccrA2) or Insertion sequence 1272 (IS1272) transposase. Further analysis identified a common IS1272 target inverted repeats (IR) sequence in ST2250 strains, providing insights into why these strains are more likely to acquire the CRISPR-Cas system. CRISPR typing identified 41 sequences types, classifying these strains into two clusters, with Cluster II being the predominant one. Homology analysis of spacers revealed that all the identified 15 spacers exhibited homology to sequences from plasmids, lytic phages, or prophages.

CONCLUSION: This study suggests that the acquisition of the CRISPR-Cas system in S. argenteus enhances its resistance to phage attacks and plasmid invasions in environmental settings, potentially posing significant challenges for clinical treatment of infections caused by these strains and hindering efforts to control their spread in food products using phage-based interventions.},
}

*CRISPR-Cas Systems/genetics
*Staphylococcus/genetics/classification/isolation & purification
Humans
*Genome, Bacterial
*Staphylococcal Infections/microbiology/epidemiology/veterinary
Animals
Genomics
DNA Transposable Elements
Prevalence
Food Microbiology
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40790872,
year = {2025},
author = {Kale, SM and Paina, C and Füchtbauer, WS and Bjarup, P and Sarup, PM and Schatz-Jakobsen, JA and Orabi, J and Borum, F and Jahoor, A and Brinch-Pedersen, H},
title = {An introgression from Triticum timopheevii reduces grain protein content in winter wheat populations.},
journal = {The plant genome},
volume = {18},
number = {3},
pages = {e70090},
doi = {10.1002/tpg2.70090},
pmid = {40790872},
issn = {1940-3372},
support = {Halm til det hele//Promilleafgiftsfonden for Landbrugs/ ; },
mesh = {*Triticum/genetics/metabolism ; Genome-Wide Association Study ; *Grain Proteins/metabolism ; Phenotype ; Quantitative Trait Loci ; Plant Breeding ; *Genetic Introgression ; Edible Grain/genetics ; Plant Proteins/genetics/metabolism ; Chromosomes, Plant/genetics ; },
abstract = {Improving grain protein content (GPC) in wheat (Triticum aestivum L.) is crucial for enhancing end-use quality and ensuring efficient nitrogen (N) utilization, thereby reducing environmental damage caused by excess N. However, progress in increasing GPC has been limited because of the strong negative correlation between GPC and grain yield (GY), as well as the scarcity of multi-location, multi-year phenotypic data. In this study, we analyzed the variation in GPC, GY, and grain protein deviation (GPD) using multi-location, multi-year phenotypic data from winter wheat varieties in Scandinavian regions. As reported previously, we observed a negative correlation between GY and GPC, with recent cultivars showing higher GY but lower GPC. Additionally, a genome-wide association study (GWAS) in two independent populations identified significant marker-trait associations (MTAs) for GPC and GPD, with key MTAs located on chromosome 2B (chr2B), highlighting its central role in the regulation of these traits. Interestingly, the MTA for GPD on chr2B coincided with an introgression from Triticum timopheevii, which was associated with reduced GPC and GPD in elite lines carrying this region. This introgression, which contains a powdery mildew resistance gene (Pm6), appears to negatively affect GPC, likely due to linkage drag. These findings emphasize the importance of chr2B in wheat breeding and suggest that advanced genomic techniques, such as mutagenesis and CRISPR-Cas, could be employed to mitigate negative pleiotropic effects and improve GPC and GPD. Overall, this study provides valuable insights into the genetic architecture underlying GPC in wheat and offers directions for future breeding strategies aimed at enhancing protein content.},
}

*Triticum/genetics/metabolism
Genome-Wide Association Study
*Grain Proteins/metabolism
Phenotype
Quantitative Trait Loci
Plant Breeding
*Genetic Introgression
Edible Grain/genetics
Plant Proteins/genetics/metabolism
Chromosomes, Plant/genetics

@article {pmid40790091,
year = {2025},
author = {Kocsy, K and Wilkinson, H and Felix-Ilemhenbhio, F and Bax, B and Van Agtmael, T and Azzouz, M and Majid, A},
title = {Gene editing for collagen disorders: current advances and future perspectives.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {40790091},
issn = {1476-5462},
abstract = {Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.},
}

@article {pmid40696172,
year = {2025},
author = {Xu, Z and Wang, G and Zhu, X and Wang, R and Zhu, L and Tu, L and Liu, Y and Peng, R and Lindsey, K and Wang, M and Zhang, X and Jin, S},
title = {Genome assembly of two allotetraploid cotton germplasms reveals mechanisms of somatic embryogenesis and enables precise genome editing.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {2028-2039},
pmid = {40696172},
issn = {1546-1718},
support = {32325039//China National Funds for Distinguished Young Scientists/ ; 32201856//National Science Foundation of China | Young Scientists Fund/ ; },
mesh = {*Gossypium/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Tetraploidy ; *Plant Somatic Embryogenesis Techniques/methods ; Retroelements/genetics ; CRISPR-Cas Systems ; },
abstract = {Somatic embryogenesis is crucial for plant genetic engineering, yet the underlying mechanisms in cotton remain poorly understood. Here we present a telomere-to-telomere assembly of Jin668 and a high-quality assembly of YZ1, two highly regenerative allotetraploid cotton germplasms. The completion of the Jin668 genome enables characterization of ~30.1 Mb of centromeric regions invaded by centromeric retrotransposon of maize and Tekay retrotransposons, an ~8.1 Mb 5S rDNA array containing 25,190 copies and a ~75.1 Mb major 45S rDNA array with 8,131 copies. Comparative analyses of regenerative and recalcitrant genotypes reveal dynamic transcriptional patterns and chromatin accessibility during the initial regeneration process. A hierarchical gene regulatory network identifies AGL15 as a contributor to regeneration. Additionally, we demonstrate that genetic variation affects sgRNA target sites, while the Jin668 genome assembly reduces the risk of off-target effects in CRISPR-based genome editing. Together, the complete Jin668 genome reveals the complexity of genomic regions and cotton regeneration, and improves the precision of genome editing.},
}

*Gossypium/genetics
*Genome, Plant/genetics
*Gene Editing/methods
Tetraploidy
*Plant Somatic Embryogenesis Techniques/methods
Retroelements/genetics
CRISPR-Cas Systems

@article {pmid40607979,
year = {2025},
author = {Cohen, O and Maru, P and Liang, Q and Saeij, JPJ},
title = {Surface antigen SAG1 mediates Toxoplasma gondii fitness and host cell attachment in IFNγ-stimulated cells.},
journal = {Infection and immunity},
volume = {93},
number = {8},
pages = {e0001025},
doi = {10.1128/iai.00010-25},
pmid = {40607979},
issn = {1098-5522},
support = {//Societe de Pathologie Infectieuse de Langue Francaise/ ; R01AI173803//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Toxoplasma/physiology/genetics/immunology ; *Interferon-gamma/immunology/pharmacology ; *Antigens, Protozoan/genetics/metabolism/immunology ; *Protozoan Proteins/genetics/metabolism/immunology ; *Host-Parasite Interactions ; Animals ; Humans ; Toxoplasmosis/parasitology/immunology ; Cell Adhesion ; CRISPR-Cas Systems ; Mice ; },
abstract = {Toxoplasma gondii is an obligate intracellular protozoan parasite that can establish lifelong infections and cause severe disease in immunocompromised individuals. Interferon gamma (IFNγ) is a key host defense cytokine that induces a variety of toxoplasmacidal mechanisms. Recent CRISPR/Cas9 loss-of-function screens identified multiple Toxoplasma genes important for fitness in IFNγ-stimulated cells. One consistent hit in several screens was the parasite surface antigen, SAG1. Here, we used CRISPR/Cas9 to generate a SAG1 knockout strain and found that SAG1 is important for parasite fitness specifically in IFNγ-stimulated cells. Mechanistic studies revealed that host surface sialic acids are important for parasite attachment, especially in IFNγ-stimulated cells. SAG1-deficient parasites had reduced attachment efficiency, which was exacerbated in IFNγ-treated cells. These findings highlight the role of SAG1 in mediating robust parasite attachment, especially in the context of immune pressure.},
}

*Toxoplasma/physiology/genetics/immunology
*Interferon-gamma/immunology/pharmacology
*Antigens, Protozoan/genetics/metabolism/immunology
*Protozoan Proteins/genetics/metabolism/immunology
*Host-Parasite Interactions
Animals
Humans
Toxoplasmosis/parasitology/immunology
Cell Adhesion
CRISPR-Cas Systems
Mice

@article {pmid40579538,
year = {2025},
author = {Hanlon, VCT and Cagan, A and Eves-van den Akker, S},
title = {How and when organisms edit their own genomes.},
journal = {Nature genetics},
volume = {57},
number = {8},
pages = {1823-1834},
pmid = {40579538},
issn = {1546-1718},
support = {BB/S006397/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/X006352/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/Y513246/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; RPG-2023-001//Leverhulme Trust/ ; R01AG087974//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Genome/genetics ; Bacteria/genetics ; Host-Pathogen Interactions/genetics ; },
abstract = {Mutations are often thought of as untargeted and non-adaptive, but in rare cases, organisms perform programmed, targeted and adaptive rearrangements of their own DNA sequences. Notable examples include the somatic diversification of immunoglobulin genes, which is the foundation of the vertebrate immune system, and natural CRISPR spacer arrays in bacteria, which recognize and cleave foreign DNA. These systems, along with a dozen known analogs scattered across the tree of life, often underlie critical biological functions, particularly in host-pathogen conflicts. In this Review, we compare the mechanisms by which organisms edit their own genomes. We show that superficially dissimilar editing systems often rely on surprisingly similar genetic mechanisms, regardless of function or taxon. Finally, we argue that the recurrence of editing in host-pathogen conflicts and the bias to a handful of well-studied organisms strongly suggest that new editing systems will be found in understudied pathogens and their hosts.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
*Genome/genetics
Bacteria/genetics
Host-Pathogen Interactions/genetics

@article {pmid40519079,
year = {2025},
author = {Geronazzo, J and Heimerl, A and Lindell, L and McCrimmon, S and Stormer, C and Horvai, B and Johnson, IP and Peterson, TM and Zuckerman, J and Scott, AI and Course, MM},
title = {Characterizing fatty acid oxidation genes in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf139},
pmid = {40519079},
issn = {2160-1836},
support = {//CC Natural Sciences Executive Committee Research and Development/ ; //CC Student Collaborative Research/ ; //Seattle Children's Research Institute Center for Clinical and Translational Research/ ; //AIS/ ; },
mesh = {Animals ; *Fatty Acids/metabolism ; Oxidation-Reduction ; *Drosophila Proteins/genetics/metabolism ; Phenotype ; *Drosophila/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; Mutation ; *Drosophila melanogaster/genetics/metabolism ; },
abstract = {In this study, we leverage the power and tractability of Drosophila genetics to better understand the molecular mechanisms underlying a group of rare genetic diseases known as fatty acid oxidation disorders. We use CRISPR-Cas9 to generate mutations in 6 putative fatty acid oxidation genes in Drosophila, then analyze the phenotypes and acylcarnitine profiles of these flies. We find that while Arc42 and CG4860 are both predicted orthologs of human ACADS, only Arc42 loss of function mirrors the acylcarnitine profile of ACADS loss of function. Acylcarnitine profiles also support our previous identification of Mcad as the likely ACADM ortholog, and reveal the deleterious effects of a single codon deletion in Mtpα (the predicted human HADHA ortholog). Finally, we observe that loss of function in Etf-QO and in CG7834-predicted orthologs of human ETFDH and ETFB, respectively-is homozygous lethal in flies. Producing animal models like these will enable new approaches to studying fatty acid oxidation disease progression, symptomatic variability, and therapeutic intervention.},
}

Animals
*Fatty Acids/metabolism
Oxidation-Reduction
*Drosophila Proteins/genetics/metabolism
Phenotype
*Drosophila/genetics/metabolism
Humans
CRISPR-Cas Systems
Mutation
*Drosophila melanogaster/genetics/metabolism

@article {pmid40476353,
year = {2025},
author = {Weisman, AS and Fisher, NM and Hunter, CP},
title = {Efficient iterative CRISPR/Cas9 editing using sid-1 co-conversion and feeding RNAi in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf128},
pmid = {40476353},
issn = {2160-1836},
support = {GM089795/NH/NIH HHS/United States ; 62579/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Caenorhabditis elegans Proteins/genetics ; *RNA Interference ; Phenotype ; Membrane Proteins ; },
abstract = {We present a sid-1 loss-of-function and restoration-of-function CRISPR/Cas9 co-conversion protocol in Caenorhabditis elegans. Introducing CRISPR reagents that induce sid-1 loss-of-function can produce survivors on lethal RNAi foods while reagents that induce sid-1 restoration-of-function can be screened for restoration of visible RNAi phenotypes. Both methods efficiently reduce the pool of candidates from hundreds or thousands of F1 progeny to tens with minimal experimenter effort. Furthermore, our optimized sid-1 CRISPR design allows a high ratio of CRISPR reagents targeting the gene of interest, maximizing successful co-conversion events. The interconvertibility of the sid-1 locus readily enables this strategy to be leveraged to iteratively create complex strains with multiple gene edits.},
}

Animals
*Caenorhabditis elegans/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Caenorhabditis elegans Proteins/genetics
*RNA Interference
Phenotype
Membrane Proteins

@article {pmid40460280,
year = {2025},
author = {Nalley, MJ and Banerjee, S and Huang, MY and Madhani, HD},
title = {Near 100% efficient homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {8},
pages = {},
doi = {10.1093/g3journal/jkaf118},
pmid = {40460280},
issn = {2160-1836},
support = {//R01 AI000272/ ; },
mesh = {*Cryptococcus neoformans/genetics ; *Genome, Fungal ; CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; DNA End-Joining Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {We recently described CRISPR/Cas9-based short homology-dependent genome engineering in the human fungal pathogen Cryptococcus neoformans, a haploid budding yeast that is the most common cause of fungal meningitis and an emerging model organism. This was achieved by electroporation of strains stably expressing a codon-optimized Cas9 with 2 separate DNA molecules, one encoding a selectable marker flanked by short homology arms and a second encoding a sgRNA under the control of the U6 snRNA promoter. However, the efficiency of desired homology-dependent repair relative to undesired non-homologous end-joining (NHEJ) events can be low and variable. Here, we describe methods and strains enabling extremely efficient (∼99%) homology-dependent genome editing in C. neoformans. This high-efficiency method requires 2 manipulations. First, we placed the sgRNA-expressing segment into the marker-containing DNA flanked by targeting homology; thus, only a single DNA molecule is introduced into cells. Second, we used a strain mutant for the non-homologous end-joining factor Ku80 (encoded by YKU80). We also report the engineering of a yku80::amdS mutant strain harboring an insertion mutation that can be removed scarlessly via recombination between direct repeats. This enables the functional restoration of YKU80 after homology-dependent genome editing after selection against the amdS marker using fluoroacetamide. This approach minimizes documented drawbacks of using Ku-defective strains in downstream experiments. Finally, we describe a plasmid series that enables rapid cloning of sgRNA-marker constructs for genomic manipulation of C. neoformans, including gene deletion and C-terminal tagging. These methods, strains, and plasmids accelerate the genomic manipulation of C. neoformans.},
}

*Cryptococcus neoformans/genetics
*Genome, Fungal
CRISPR-Cas Systems
*Gene Editing/methods
Humans
DNA End-Joining Repair
*Genetic Engineering/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40188316,
year = {2025},
author = {Teter, OM and McQuade, A and Hagan, V and Liang, W and Dräger, NM and Sattler, SM and Holmes, BB and Castillo, VC and Papakis, V and Leng, K and Boggess, S and Nowakowski, TJ and Wells, J and Kampmann, M},
title = {CRISPRi-based screen of autism spectrum disorder risk genes in microglia uncovers roles of ADNP in microglia endocytosis and synaptic pruning.},
journal = {Molecular psychiatry},
volume = {30},
number = {9},
pages = {4176-4193},
pmid = {40188316},
issn = {1476-5578},
support = {U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; 2034836//National Science Foundation (NSF)/ ; AARF-22-973222//Alzheimer's Association/ ; 2022-A-016-FEL//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; U01 MH115747/MH/NIMH NIH HHS/United States ; R01 MH125516/MH/NIMH NIH HHS/United States ; },
mesh = {*Microglia/metabolism ; *Autism Spectrum Disorder/genetics/metabolism ; Humans ; Endocytosis/genetics/physiology ; Phagocytosis/genetics ; Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Neuronal Plasticity/genetics/physiology ; CRISPR-Cas Systems/genetics ; Coculture Techniques ; *Nerve Tissue Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synapses/metabolism ; Genetic Predisposition to Disease/genetics ; Brain/metabolism ; },
abstract = {Autism Spectrum Disorders (ASD) are a set of neurodevelopmental disorders with complex biology. The identification of ASD risk genes from exome-wide association studies and de novo variation analyses has enabled mechanistic investigations into how ASD-risk genes alter development. Most functional genomics studies have focused on the role of these genes in neurons and neural progenitor cells. However, roles for ASD risk genes in other cell types are largely uncharacterized. There is evidence from postmortem tissue that microglia, the resident immune cells of the brain, appear activated in ASD. Here, we used CRISPRi-based functional genomics to systematically assess the impact of ASD risk gene knockdown on microglia activation and phagocytosis. We developed an iPSC-derived microglia-neuron coculture system and high-throughput flow cytometry readout for synaptic pruning to enable parallel CRISPRi-based screening of phagocytosis of beads, synaptosomes, and synaptic pruning. Our screen identified ADNP, a high-confidence ASD risk genes, as a modifier of microglial synaptic pruning. We found that microglia with ADNP loss have altered endocytic trafficking, remodeled proteomes, and increased motility in coculture.},
}

*Microglia/metabolism
*Autism Spectrum Disorder/genetics/metabolism
Humans
Endocytosis/genetics/physiology
Phagocytosis/genetics
Neurons/metabolism
Induced Pluripotent Stem Cells/metabolism
*Neuronal Plasticity/genetics/physiology
CRISPR-Cas Systems/genetics
Coculture Techniques
*Nerve Tissue Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Synapses/metabolism
Genetic Predisposition to Disease/genetics
Brain/metabolism

@article {pmid39394482,
year = {2025},
author = {Mantena, S and Pillai, PP and Petros, BA and Welch, NL and Myhrvold, C and Sabeti, PC and Metsky, HC},
title = {Model-directed generation of artificial CRISPR-Cas13a guide RNA sequences improves nucleic acid detection.},
journal = {Nature biotechnology},
volume = {43},
number = {8},
pages = {1266-1273},
pmid = {39394482},
issn = {1546-1696},
support = {U01AI151812//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; T32 GM144273/GM/NIGMS NIH HHS/United States ; T32GM007753//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32GM144273//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; U19AI110818//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; D18AC00006//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; K01AI163498//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; NU50CK000629//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; INV-034761/GATES/Gates Foundation/United States ; INV-034761/GATES/Gates Foundation/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Algorithms ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR guide RNA sequences deriving exactly from natural sequences may not perform optimally in every application. Here we implement and evaluate algorithms for designing maximally fit, artificial CRISPR-Cas13a guides with multiple mismatches to natural sequences that are tailored for diagnostic applications. These guides offer more sensitive detection of diverse pathogens and discrimination of pathogen variants compared with guides derived directly from natural sequences and illuminate design principles that broaden Cas13a targeting.},
}

*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Algorithms
Base Sequence
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40790054,
year = {2025},
author = {Patel, MA and Boribong, BP and Sinha, H and Xiao, B and Xie, K and Vo, PQN and Chin, AB and Ellouzi, A and Little, SR and Shih, S and Wu, H and Muller, WJ and Hirukawa, A},
title = {Miniaturized scalable arrayed CRISPR screening in primary cells enables discovery at the single donor resolution.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {29350},
pmid = {40790054},
issn = {2045-2322},
mesh = {Humans ; *CRISPR-Cas Systems ; Electroporation/methods ; *Gene Editing/methods ; Microfluidics/methods ; CD4-Positive T-Lymphocytes/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Miniaturization ; },
abstract = {High-efficiency gene editing in primary human cells is critical for advancing therapeutic development and functional genomics, yet conventional electroporation platforms often require high cell input and are poorly suited to parallelized experiments. Here we introduce a next-generation digital microfluidics (DMF) electroporation platform that enables high-throughput, low-input genome engineering using discrete droplets manipulated on a planar electrode array. The system supports 48 independently programmable reaction sites and integrates seamlessly with laboratory automation, allowing efficient delivery of CRISPR-Cas9 RNPs and mRNA cargo into as few as 3,000 primary human cells per condition. The platform was validated across diverse primary human cell types and cargo modalities, demonstrating efficient delivery of various cargo, with high rates of transfection, gene knockout via non-homologous end joining, and precise knock-in through homology-directed repair. To showcase its utility in functional genomics, we applied the platform to an arrayed CRISPR-Cas9 screen in chronically stimulated human CD4[+] T cells, identifying novel regulators of exhaustion, including epigenetic and transcriptional modulators. These findings establish our DMF-based electroporation platform as a powerful tool for miniaturized genome engineering in rare or precious cell populations and provide a scalable framework for high-content genetic screening in primary human cells.},
}

Humans
*CRISPR-Cas Systems
Electroporation/methods
*Gene Editing/methods
Microfluidics/methods
CD4-Positive T-Lymphocytes/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Miniaturization

@article {pmid40790042,
year = {2025},
author = {Li, H and He, Y and Jiang, J and Liu, Z and Liu, Y and Shi, Q and Ding, J and Li, H and Sun, W and Hu, X and Chen, Z and He, X},
title = {CRISPR screening reveals that RNA helicase DDX41 triggers ribosome biogenesis and cancer progression through R-loop-mediated RPL/RPS transcription.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7409},
pmid = {40790042},
issn = {2041-1723},
support = {82121004//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472626//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82472641//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82172937//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*DEAD-box RNA Helicases/genetics/metabolism ; Humans ; *Ribosomes/metabolism/genetics ; Animals ; Mice ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *Ribosomal Proteins/genetics/metabolism ; Transcription, Genetic ; Disease Progression ; Histone Acetyltransferases/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Neoplasms/genetics/pathology ; Protein Biosynthesis ; },
abstract = {The RNA helicase DDX41 is a DEAD-box helicase that is well known as a virus sensor in dendritic cells and a tumor suppressor that is frequently mutated in myeloid neoplasms. However, the functions and relevance of DDX41 in solid tumors remain largely unexplored. In this study, through in vivo CRISPR screening, we demonstrate that DDX41 is highly expressed in various solid tumor types and promotes tumorigenicity in liver cancer. Mechanistically, DDX41 facilitates R-loop processing and accelerates the transcription of RPL/RPS genes, thereby promoting ribosome biogenesis and protein synthesis. Additionally, we show that the acetyltransferase KAT8 is required for H3K9ac modification of the DDX41 promoter and that NR2C1/NR2C2 are responsible for DDX41 expression. Moreover, elevated DDX41 levels increase liver cancer cell sensitivity to protein synthesis inhibitors; treatment with homoharringtonine (HHT), an approved drug, significantly inhibits tumor growth in DDX41-overexpressing liver cancer models. Taken together, the results of this study highlight that DDX41 acts as an oncogene in liver cancer and suggest that protein synthesis inhibition may be a promising therapy for liver cancers with high DDX41 expression.},
}

*DEAD-box RNA Helicases/genetics/metabolism
Humans
*Ribosomes/metabolism/genetics
Animals
Mice
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*Ribosomal Proteins/genetics/metabolism
Transcription, Genetic
Disease Progression
Histone Acetyltransferases/metabolism
Promoter Regions, Genetic
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Neoplasms/genetics/pathology
Protein Biosynthesis

@article {pmid40790018,
year = {2025},
author = {Wang, K and Wang, J and Yang, X and Sun, W and Sheng, G and Wang, Y},
title = {Structural insights into Type II-D Cas9 and its robust cleavage activity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7396},
pmid = {40790018},
issn = {2041-1723},
support = {Z231100007223004//Beijing Municipal Science and Technology Commission/ ; 32330055, 31930065, 22121003, 32071198, 32071444//National Natural Science Foundation of China (National Science Foundation of China)/ ; 5232022//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; XDB0570000, XDB37010202//Chinese Academy of Sciences (CAS)/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/genetics/ultrastructure ; Cryoelectron Microscopy ; DNA/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; DNA Cleavage ; CRISPR-Cas Systems ; Gene Editing ; *Bacterial Proteins/metabolism/genetics/chemistry ; Catalytic Domain ; },
abstract = {Type II-D Cas9 proteins (Cas9d) are more compact than typical Type II-A/B/C Cas9s. Here, we demonstrate that NsCas9d from Nitrospirae bacterium RBG_13_39_12 derived from a metagenomic assembly exhibits robust dsDNA cleavage activity comparable to SpCas9 in vitro. Unlike typical Cas9 enzymes that generate blunt ends, NsCas9d produces 3-nucleotide staggered overhangs. Our high-resolution cryo-EM structure of the NsCas9d-sgRNA-dsDNA complex in its catalytic state reveals the target and non-target DNA strands positioned within the HNH and RuvC catalytic pockets, respectively. NsCas9d recognizes the 5'-NRG-3' protospacer adjacent motif (PAM), with 5'-NGG-3' showing the highest cleavage efficiency. Its sgRNA structure, resembling the 5' end of IscB ωRNA, along with structural features shared with other Cas9 variants, suggests that Cas9d are hypothesized to resemble evolutionary intermediates between other Cas9 sub-types and IscB. These findings deepen our understanding of Cas9 evolution and mechanisms, highlighting NsCas9d as a promising genome-editing tool due to its compact size, DNA cleavage pattern, and efficient PAM recognition.},
}

*CRISPR-Associated Protein 9/metabolism/chemistry/genetics/ultrastructure
Cryoelectron Microscopy
DNA/metabolism/chemistry/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
DNA Cleavage
CRISPR-Cas Systems
Gene Editing
*Bacterial Proteins/metabolism/genetics/chemistry
Catalytic Domain

@article {pmid40789840,
year = {2025},
author = {Goyal, H and Kaur, J},
title = {Long non-coding RNAs and autophagy: dual drivers of Hepatocellular carcinoma progression.},
journal = {Cell death discovery},
volume = {11},
number = {1},
pages = {376},
pmid = {40789840},
issn = {2058-7716},
abstract = {Hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality worldwide, is characterized by poor prognosis, high recurrence rates, and limited responsiveness to current therapies. Autophagy, a conserved catabolic pathway essential for cellular homeostasis, plays a paradoxical role in HCC, acting as a tumor suppressor during initiation but promoting survival and progression in advanced stages. Long non-coding RNAs (lncRNAs) have emerged as critical regulators of autophagy, influencing tumorigenesis, metastasis, and therapy resistance through mechanisms such as miRNA sponging, chromatin remodeling, and protein interactions. This review describes how autophagy contributes to HCC at different stages, outlines the dual functions of lncRNAs as oncogenic drivers or tumor suppressors, and illustrates their integration into key signaling networks of autophagy (e.g., PI3K/AKT/mTOR, AMPK, Beclin-1). LncRNAs have been shown to modulate drug resistance, including resistance to first-line agents, by altering autophagic flux and associated molecular pathways. We also explored emerging strategies for targeting the lncRNA-autophagy axis, such as siRNAs, antisense oligonucleotides, and CRISPR/Cas systems, that have shown promise in preclinical studies and may be adapted for HCC. Furthermore, autophagy-related lncRNAs hold potential as non-invasive diagnostic and prognostic biomarkers and as predictors of recurrence. Integrating multi-omics approaches to validate these candidates will be critical for translation into clinical practice. Collectively, this review highlights the lncRNA-autophagy network as a promising frontier of biomarker discovery for precision diagnostics and targets for innovative therapeutics. The regulatory role of lncRNAs in autophagy presents a paradigm shift, heralding new strategies for targeted treatment.},
}

@article {pmid40789802,
year = {2025},
author = {Noormohamadi, H and Soleimani Samarkhazan, H and Kargar, M and Maroufi, F and Servatian, N and Davami, F},
title = {CRISPR/Cas technologies in pancreatic cancer research and therapeutics: recent advances and future outlook.},
journal = {Discover oncology},
volume = {16},
number = {1},
pages = {1530},
pmid = {40789802},
issn = {2730-6011},
abstract = {Pancreatic cancer is marked by a poor prognosis and an exceptionally high mortality rate, with its aggressive nature contributing to its classification as a highly malignant disease. For effective therapeutic strategies, the development of sophisticated and regulated DNA manipulation methods is essential. Originally part of the prokaryotic immune system, CRISPR/Cas has emerged as a pivotal genome-editing tool with promising applications in pancreatic cancer research and therapy. This gene editing method is known for simplicity, rapid advancement, and superior precision compared to earlier techniques. Its adaptability allows precise gene editing for therapeutic purposes, including oncogene silencing and correction of pathogenic mutations. Additionally, CRISPR-driven gene editing has facilitated the development of pancreatic cancer models, which serve as valuable platforms for drug discovery and personalized treatment strategies, offering deeper insights into the genetic landscape of pancreatic tumors. This article provides an overview of the current applications of CRISPR technology in gene therapy and cancer research, particularly in the context of pancreatic cancer, and lays the foundation for future studies.},
}

@article {pmid40789027,
year = {2025},
author = {Huang, Y and Li, C and Desingu Rajan, AR and Bronner, ME},
title = {Sox11 genes affect neuronal differentiation in the developing zebrafish enteric nervous system.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {33},
pages = {e2510548122},
doi = {10.1073/pnas.2510548122},
pmid = {40789027},
issn = {1091-6490},
support = {HD105604//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; DK133480//HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Enteric Nervous System/embryology/metabolism/cytology ; *Zebrafish Proteins/genetics/metabolism ; *SOXC Transcription Factors/genetics/metabolism ; *Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Neural Crest/metabolism/cytology ; *Neurons/metabolism/cytology ; CRISPR-Cas Systems ; Neurogenesis/genetics ; },
abstract = {The vertebrate enteric nervous system (ENS) is derived from vagal neural crest cells, which enter the foregut as progenitors that migrate from rostral to caudal to populate the entire length of the gut. Here, we show that transcription factors sox11a and sox11b, zebrafish orthologs of the human SOX11 gene, are highly expressed in neural crest cells transitioning from progenitors to differentiating neuronal subtypes. Accordingly, CRISPR-Cas9 depletion shows that loss of sox11 paralogs reduces the number of neurons that express the inhibitory motor neuron marker adcyap1b without affecting cell proliferation or death. Transcription factor footprinting analysis of open chromatin regions identified by ATAC-seq reveals Sox11 binding sites in the adcyap1b enhancer. Furthermore, mutational analysis shows these binding sites are required for mediating enhancer-driven reporter expression. Taken together, our results demonstrate an important and previously unrecognized role for sox11a and sox11b in neuronal subtype specification in the developing zebrafish ENS.},
}

Animals
*Zebrafish/genetics/embryology
*Enteric Nervous System/embryology/metabolism/cytology
*Zebrafish Proteins/genetics/metabolism
*SOXC Transcription Factors/genetics/metabolism
*Cell Differentiation/genetics
Gene Expression Regulation, Developmental
Neural Crest/metabolism/cytology
*Neurons/metabolism/cytology
CRISPR-Cas Systems
Neurogenesis/genetics

@article {pmid40788907,
year = {2025},
author = {Zhang, Y and Fu, Z and Yang, B and Wang, J and Lu, T and Shi, DL and Shao, M},
title = {Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {221},
pages = {},
doi = {10.3791/68642},
pmid = {40788907},
issn = {1940-087X},
mesh = {Animals ; *Zebrafish/genetics ; Female ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems ; Animals, Genetically Modified ; *RNA-Binding Proteins/genetics ; *Zebrafish Proteins/genetics ; },
abstract = {Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model.},
}

Animals
*Zebrafish/genetics
Female
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems
Animals, Genetically Modified
*RNA-Binding Proteins/genetics
*Zebrafish Proteins/genetics

@article {pmid40788566,
year = {2025},
author = {Risse, J and Pietrek, L and Cantz, T and Krzemien, M and Schnalke, J and Eggenschwiler, R and Heinemann, T and Dederer, HG},
title = {"Snip, snip, cure"? Philosophical, legal and biomedical perspectives on novel somatic genomic therapies.},
journal = {Medicine, health care, and philosophy},
volume = {},
number = {},
pages = {},
pmid = {40788566},
issn = {1572-8633},
abstract = {The advent of innovative techniques, such as the CRISPR/Cas system, has opened up a new range of possibilities for modifying the genome, with the potential to address previously unmet therapeutic needs of patients with genetic diseases. These new possibilities have not only raised ethical concerns but also challenged existing classifications of genome modification techniques. While the legal status of some of these new therapies remains uncertain, there is an ongoing debate within philosophy of biology about the information-related metaphors adopted by scientists to describe and classify the genome and its therapeutic modification. Given the continuing advance of new genomic therapies, we show, employing an interdisciplinary approach, that a comprehensive framework for the classification of these technologies is needed to resolve legal and philosophical issues. The first section provides an analysis of the current state of novel genome-modifying techniques in medical genetics. In the second section, we assess the regulatory status of these techniques within the European regulatory framework for advanced therapy medicinal products (ATMPs). Drawing on these results, we argue in the third section from a philosophical perspective that metaphors, such as 'editing' the genome, which are based on a conception of the genome as linear information, cannot adequately capture the breadth of advanced genomic technologies. To accurately categorise these techniques in a manner that meets their diverse applications, we propose introducing the umbrella term 'somatic genomic therapies' (SGTs). Urging an integrative approach to defining and classifying new technologies in medical genetics, we advocate for the development of an integrative concept of SGTs.},
}

@article {pmid40787451,
year = {2025},
author = {Sprissler, J and Pannicke, U and Rump, EM and Schrezenmeier, H and Casadei, N and Pogoda, M and Kuhlburger, L and Oquendo, MB and Czemmel, S and Debatin, KM and Erlacher, M and Schwarz, K and Felgentreff, K},
title = {RAG recombinase expression discriminates the development of natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1607664},
pmid = {40787451},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/cytology/metabolism ; Humans ; *Cell Differentiation/immunology/genetics ; Induced Pluripotent Stem Cells/immunology/metabolism/cytology ; *Homeodomain Proteins/genetics/metabolism ; V(D)J Recombination ; *DNA-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: V(D)J recombination, initiated by recombination-activating gene (RAG) endonucleases, is a crucial process for the generation of diversified antigen receptors of T and B lymphocytes but regarded dispensable for innate natural killer (NK) lymphocytes lacking clonotypic receptors.

METHODS: To explore the impact of potential rearrangements on NK cell maturation, RAG-fate mapping reporter human induced pluripotent stem cell (iPSC) lines were generated by introduction of RSS-invEGFP constructs into the AAVS1 locus using CRISPR/Cas9 and differentiated into NK cells in vitro.

RESULTS: GFP expression was observed in up to 14% of mature NK cells characterized by a CD45[dim] CD56[dim]CD57[+]NKG2C[+/-]KIR[+/-] phenotype and unproductive genetic rearrangements in the IGH locus. Advanced maturation was further revealed by transcriptomic studies using RNA sequencing. Despite their strong effector function, DNA damage response and survival to ionizing radiation were compromised.

DISCUSSION: These findings suggest a role of RAG expression in NK cell ontogeny supporting the development of a terminally differentiated effector population.},
}

*Killer Cells, Natural/immunology/cytology/metabolism
Humans
*Cell Differentiation/immunology/genetics
Induced Pluripotent Stem Cells/immunology/metabolism/cytology
*Homeodomain Proteins/genetics/metabolism
V(D)J Recombination
*DNA-Binding Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40784966,
year = {2025},
author = {Wuttinontananchai, C and Yamamoto, J and Sakamoto, S and Yamaguchi, Y},
title = {Genome-wide CRISPR screen for human factors involved in alternative polyadenylation based on differential localization of CD47.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {29269},
pmid = {40784966},
issn = {2045-2322},
support = {23K07807//Japan Society for the Promotion of Science/ ; 20H03182//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Polyadenylation/genetics ; *CD47 Antigen/metabolism/genetics ; *CRISPR-Cas Systems ; Genome, Human ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {At least 70% of the human protein-coding genes contain multiple polyadenylation sites (PAS) and undergo alternative polyadenylation (APA), generating distinct transcripts from a single gene. While APA has been implicated in various physiological and pathological processes, its regulatory factors and cellular mechanisms remain incompletely understood. A previous study demonstrated that APA influences the localization of the cell surface marker CD47. Here, we present the results of a genome-wide CRISPR screen aimed at identifying APA regulators using CD47 as a reporter. Given that isoform-specific knockdown of CD47, as well as knockdown of core 3' end processing factors, alters CD47 localization, we developed an immunofluorescence-based method that simultaneously detects cell surface and intracellular CD47 protein, enabling the visualization of APA-dependent changes at the single-cell level. Leveraging this approach, we conducted a CRISPR screen and identified multiple genes affecting CD47 cell-surface expression. In addition to known membrane trafficking factors, we uncovered several nuclear factors, among which POLDIP2 emerged as a potential novel APA regulator with a global impact on APA. This study provides a foundation for further investigations into the molecular mechanisms governing APA.},
}

Humans
*Polyadenylation/genetics
*CD47 Antigen/metabolism/genetics
*CRISPR-Cas Systems
Genome, Human
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40752775,
year = {2025},
author = {Meshram, HK and Gupta, SK and Gupta, A and Nagori, K and Ajazuddin, },
title = {Next-generation CRISPR gene editing tools in the precision treatment of Alzheimer's and Parkinson's disease.},
journal = {Ageing research reviews},
volume = {111},
number = {},
pages = {102851},
doi = {10.1016/j.arr.2025.102851},
pmid = {40752775},
issn = {1872-9649},
mesh = {Humans ; *Gene Editing/methods ; *Parkinson Disease/therapy/genetics ; *Alzheimer Disease/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Precision Medicine/methods ; },
abstract = {Emerging gene-editing technologies, such as the CRISPR system, represent a potential pathway for precision medicine targeting the genetic and molecular causes of diseases. Second-generation CRISPR technologies, including base editing, prime editing, and engineered Cas variants, have improved fidelity and offer alternative strategies for precise gene correction, transcriptional repression or activation, and modulation of pathological pathways in neurodegeneration. These tools can correct single-nucleotide mutations, reduce pathological protein accumulation, and modulate neuroinflammatory responses, all integral to the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD), both chronic, progressive neurodegenerative disorders. Unfortunately, currently available treatments are limited and primarily palliative. Preclinical studies have shown promising results, with improvements in cognitive and motor deficits in animal models. However, significant challenges must be addressed to ensure safe and effective delivery to the CNS, minimize off-target effects, and address ethical concerns. Current clinical investigations aim to translate these findings into available therapeutic options. This review also identifies the biological mechanisms, therapeutic use cases, and current limitations of next-generation CRISPR systems as tools in the context of AD and PD, providing both therapeutic and research capabilities through their unique strengths. Ultimately, the future of transactional neurogenomics will determine the clinical possibilities of CRISPR-based strategies for advancing neurodegenerative disease management beyond palliative and symptomatic treatment, toward a feasible mechanistic form of disease modification.},
}

Humans
*Gene Editing/methods
*Parkinson Disease/therapy/genetics
*Alzheimer Disease/therapy/genetics
*CRISPR-Cas Systems/genetics
Animals
*Genetic Therapy/methods
*Precision Medicine/methods

@article {pmid40729503,
year = {2025},
author = {Bao, B and Li, L and Li, M and Long, Y and Zhu, Y and Yin, J and Zhang, X and Tang, Y},
title = {Biomimetic Composite Nanoparticles with Immune Modulation and CRISPR Gene Editing for Enhancing Mild Photothermal Therapy-Based Synergistic Antitumor Therapy.},
journal = {Biomacromolecules},
volume = {26},
number = {8},
pages = {5245-5257},
doi = {10.1021/acs.biomac.5c00735},
pmid = {40729503},
issn = {1526-4602},
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Photothermal Therapy/methods ; Female ; *Gene Editing/methods ; *Biomimetic Materials/chemistry/pharmacology ; Humans ; Cell Line, Tumor ; Mice, Inbred BALB C ; HSP90 Heat-Shock Proteins/genetics ; CRISPR-Cas Systems ; *Breast Neoplasms/therapy ; Tumor Microenvironment/drug effects ; RAW 264.7 Cells ; },
abstract = {Photothermal therapy (PTT) is a promising cancer treatment. However, the high temperature generated during therapy may harm normal tissues, and the immunosuppressive microenvironment induced by tumor-associated macrophages (TAMs) hinders immune clearance of residual tumors after PTT. Therefore, developing mild PTT and remodeling the immunosuppressive microenvironment are crucial to improve antitumor efficacy and irradiation safety. Herein, we developed a biomimetic composite nanoparticle based on ribonucleoprotein (RNP), tetra-methylphenidine (TMP195), and a designed amphiphilic NIR-II conjugated polymer, PCQ-PEG-NH2. PCQ-PEG-NH2 exhibits a high photothermal conversion efficiency (58.1%), enabling PTT under safe laser intensity. The loaded RNP specifically knocks down the HSP90α gene, reducing tumor thermotolerance to enhance mild PTT efficiency. TMP195 reprograms TAMs from M2 to M1 phenotype, alleviating immunosuppression. Additionally, macrophage membrane modification endows nanoparticles with excellent biocompatibility and active tumor-targeting ability. In a breast cancer mouse model, this synergistic strategy outperformed traditional PTT, providing a promising strategy for mild-PTT tumor therapy with high efficacy.},
}

Animals
*Nanoparticles/chemistry
Mice
*Photothermal Therapy/methods
Female
*Gene Editing/methods
*Biomimetic Materials/chemistry/pharmacology
Humans
Cell Line, Tumor
Mice, Inbred BALB C
HSP90 Heat-Shock Proteins/genetics
CRISPR-Cas Systems
*Breast Neoplasms/therapy
Tumor Microenvironment/drug effects
RAW 264.7 Cells

@article {pmid40651704,
year = {2025},
author = {Zhao, B and Shi, J and Zhao, R and Gao, S and Li, Y and Zhang, Y and Wei, Y and Guo, Y},
title = {Constructing CRISPR-Cas9 system for metabolic reprogramming and cordycepin biomanufacturing in Pichia pastoris.},
journal = {Bioresource technology},
volume = {436},
number = {},
pages = {132967},
doi = {10.1016/j.biortech.2025.132967},
pmid = {40651704},
issn = {1873-2976},
mesh = {*CRISPR-Cas Systems/genetics ; *Deoxyadenosines/biosynthesis/metabolism ; *Metabolic Engineering/methods ; Fermentation ; Bioreactors ; *Saccharomycetales/metabolism/genetics ; Metabolic Reprogramming ; },
abstract = {Cordycepin, a nucleoside analog mainly produced byCordyceps militaris, is widely used in food, medicine, and feed industries.Conventional microbial engineering faces challenges from antibiotic resistance genes, which increase environmental risks.Here, we engineeredPichia pastorisusing an optimized CRISPR-Cas9 system with gRNA-tRNA array and Brex27-enhanced homologous recombination, achieving antibiotic marker-free cordycepin biosynthesis. Through modular metabolic engineering strategies that optimized promoter combinations, gene copy numbers, methanol assimilation, precursor supply, and ATP/NADPH balance, strain PC19 achieved 2509.7 mg/L cordycepin in shake-flask fermentation. In fed-batch fermentation, PC19 achieved the highest production of 18.3 g/L (3.05 g/L/d and 122.2 mg/g DCW) to date in a 10 L bioreactor, and the CO2-eq emissions were 3.3-57.6 times lower than C. militaris and other microbial cell factories. This CRISPR-Cas9 system lays the foundation for low-carbon and efficient biosynthesis of cordycepin and other nucleoside analogs inP. pastoris.},
}

*CRISPR-Cas Systems/genetics
*Deoxyadenosines/biosynthesis/metabolism
*Metabolic Engineering/methods
Fermentation
Bioreactors
*Saccharomycetales/metabolism/genetics
Metabolic Reprogramming

@article {pmid40627934,
year = {2026},
author = {Guo, A and Wu, Y and Xie, Y and Guo, W and Guo, Y and Zhang, X and Zhang, W and Zou, X and Sun, Z},
title = {CRISPR-based fluorescent aptasensor combined with smartphone for on-site visual detection of DEHP in packaged foods.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {344},
number = {Pt 1},
pages = {126649},
doi = {10.1016/j.saa.2025.126649},
pmid = {40627934},
issn = {1873-3557},
mesh = {*Smartphone ; *Diethylhexyl Phthalate/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *Food Contamination/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; *CRISPR-Cas Systems ; *Food Packaging ; Spectrometry, Fluorescence/methods ; Fluorescent Dyes/chemistry ; *Food Analysis/methods ; },
abstract = {Di-(2-ethylhexyl) phthalate (DEHP) can leach into food and the environment, posing health and ecological risks. This paper introduces a novel CRISPR-based fluorescent aptasensor for on-site DEHP detection. The aptasensor selectively binds to DEHP, triggering a competitive displacement reaction that releases an Aptamer-dsDNA probe. After magnetic separation, the supernatant is analyzed via a Cas12a reporter system, where Cas12a activation cleaves a FAM-BHQ reporter, generating fluorescence. Combined with smartphone imaging, this method enables rapid result acquisition. The aptasensor shows high selectivity and sensitivity, detecting DEHP from 1 pg/mL to 1 μg/mL, with a lower limit of 0.15 pg/mL. It effectively detects DEHP in various real samples, offering reliable visual monitoring. This method offers a rapid and effective on-site detection strategy for food safety and environmental pollution monitoring.},
}

*Smartphone
*Diethylhexyl Phthalate/analysis
*Aptamers, Nucleotide/chemistry/genetics
*Food Contamination/analysis
*Biosensing Techniques/methods
Limit of Detection
*CRISPR-Cas Systems
*Food Packaging
Spectrometry, Fluorescence/methods
Fluorescent Dyes/chemistry
*Food Analysis/methods

@article {pmid40783509,
year = {2025},
author = {Luo, X and Dou, Y and Lang, Y and Zhao, H and Liu, X and Zhang, X and Li, Y and Liang, D and Xia, H},
title = {CRISPR/Cas9-mediated editing of carotenoid biosynthesis genes alters carotenoid concentrations in kiwifruit.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1056},
pmid = {40783509},
issn = {1471-2229},
support = {32472679//National Natural Science Foundation of China/ ; 2024-YF05-00408-SN//Chengdu Science and Technology Department/ ; 2023YFN0095, 2024JDRC0011, 2025ZNSFSC0190//Sichuan Province Science and Technology Department Projects/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Actinidia/genetics/metabolism ; *Carotenoids/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Fruit/genetics/metabolism ; Genes, Plant ; Agrobacterium/genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 technology has garnered increasing attention for its simplicity and precision in genome editing, making it an indispensable tool for gene function research and crop genetic improvement. However, the inefficiency and time-consuming nature of genetic transformation continue to pose substantial challenges to its widespread application in woody plants.

RESULTS: In this study, we developed a rapid and efficient Agrobacterium-mediated transformation system using petioles as explants for kiwifruit. Positive resistant seedlings were obtained within three months by inoculating on MS medium supplemented with 2.0 mg·L[-1] 6-benzylaminopurine (6-BA), 0.2 mg·L[-1] naphthaleneacetic acid (NAA), and 10 mg·L[-1] hygromycin, which was faster than using leaves as explants. Using this system, CRISPR/Cas9-mediated editing of phytoene desaturase (AcPDS) and ζ-carotene desaturase (AcZDS) achieved an editing efficiency of 20%. Transgenic kiwifruit lines with edited AcZDS exhibited a significant reduction in carotenoid content.

CONCLUSIONS: Overall, we established an efficient Agrobacterium-mediated transformation system using petioles as explants, which is applicable for CRISPR/Cas9-mediated gene editing in kiwifruit, thereby facilitating functional gene studies and genetic improvement.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Actinidia/genetics/metabolism
*Carotenoids/metabolism
Plants, Genetically Modified/genetics/metabolism
Oxidoreductases/genetics/metabolism
Fruit/genetics/metabolism
Genes, Plant
Agrobacterium/genetics

@article {pmid40779487,
year = {2025},
author = {Polinski, NK and Puoliväli, J and Rauhala, L and Stenius, TK and Bragge, T and Parkkari, T and Penttinen, AM and Chen, Y and Mabrouk, OS and Glajch, KE and Hirst, WD and Perkinton, M and Martinez, TN and Frasier, MA},
title = {Expression of human A53T alpha-synuclein without endogenous rat alpha-synuclein fails to elicit Parkinson's disease-related phenotypes in a novel humanized rat model.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329823},
pmid = {40779487},
issn = {1932-6203},
mesh = {Animals ; *alpha-Synuclein/genetics/metabolism ; Humans ; *Parkinson Disease/genetics/pathology/metabolism ; Rats ; Disease Models, Animal ; Phenotype ; Rats, Sprague-Dawley ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Male ; Rats, Transgenic ; },
abstract = {Alpha-synuclein (aSyn) is linked to Parkinson's disease (PD) through SNCA genetic mutations, phosphorylated aSyn in Lewy bodies and Lewy neurites, and most recently through evidence of aSyn aggregation in patient spinal fluid using the aSyn seed amplification assay. Therefore, understanding the biology of this protein and developing therapeutic interventions targeting pathological processing of aSyn are a key area of focus for novel treatments to slow or stop PD. Reliable preclinical models are imperative for these efforts. To this end, we developed a novel model using CRISPR/Cas9 to humanize the regions surrounding the naturally occurring threonine 53 amino acid in the Sprague Dawley rat to generate a humanized A53T aSyn rat model (aSyn A53T KI). We also generated an Snca knockout (aSyn KO) line to pair with the humanized A53T aSyn rat line to confirm that phenotypes were not due to loss of endogenous rat aSyn protein. A systematic phenotyping study was performed on these lines, assessing PD-related pathology and phenotypes at multiple timepoints. The aSyn KO rat line was profiled at 6 and 12 months of age, revealing successful aSyn protein knockout. The aSyn A53T KI model was profiled at 4, 8, 12, and 18 months of age for motor and non-motor phenotypes, nigrostriatal degeneration, and brain pathology. We confirmed the aSyn A53T KI rat expresses human aSyn while lacking endogenous rat aSyn. Motor function and non-motor function remain largely unaffected in this model, and no overt nigrostriatal degeneration or brain pathology are observed up to 18 months of age. Although the aSyn A53T KI rat lacks the ability to model PD pathology and phenotypes at baseline, it is an ideal model for investigating the impact of exogenous synuclein aggregates or environmental triggers on human aSyn in an in vivo model system.},
}

Animals
*alpha-Synuclein/genetics/metabolism
Humans
*Parkinson Disease/genetics/pathology/metabolism
Rats
Disease Models, Animal
Phenotype
Rats, Sprague-Dawley
Gene Knockout Techniques
CRISPR-Cas Systems
Male
Rats, Transgenic

@article {pmid40651547,
year = {2025},
author = {Lu, Y and Stoof, J and Tanoé, YR and Walsh, N and Bijlsma, MF and Lei, H and Chen, T and Grahovac, J and Grützmann, R and Pilarsky, C},
title = {CRISPR/Cas9 genome engineering in PDAC: From preclinical studies to translation and clinical research.},
journal = {Seminars in cancer biology},
volume = {114},
number = {},
pages = {242-255},
doi = {10.1016/j.semcancer.2025.07.004},
pmid = {40651547},
issn = {1096-3650},
mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Carcinoma, Pancreatic Ductal/genetics/therapy/pathology ; *Pancreatic Neoplasms/genetics/therapy/pathology ; *Gene Editing/methods ; Translational Research, Biomedical ; Disease Models, Animal ; Genetic Engineering/methods ; Mice ; },
abstract = {CRISPR/Cas9 technology has emerged as a powerful tool in pancreatic ductal adenocarcinoma cancer (PDAC) research, facilitating the study of genes involved in cell signaling pathways, proliferation, migration, invasion, and chemotherapy resistance. In this review, we discuss the evolution of CRISPR technologies from sophisticated editing techniques to broad screening methods, examine the utility of isogenic models and genetically engineered mouse models (GEMMs). We also explore how CRISPR/Cas9 screens can reveal immune-tumor cell interactions, highlighting the multifaceted role of this technology in PDAC research. Moreover, we emphasize the use of CRISPR technology in diagnostics for CAR-T cell therapies, where CRISPR/Cas9 enhances the precision of targeting malignant cells while minimizing off-tumor effects.},
}

*CRISPR-Cas Systems
Humans
Animals
*Carcinoma, Pancreatic Ductal/genetics/therapy/pathology
*Pancreatic Neoplasms/genetics/therapy/pathology
*Gene Editing/methods
Translational Research, Biomedical
Disease Models, Animal
Genetic Engineering/methods
Mice

@article {pmid40527655,
year = {2025},
author = {Momokawa, N and Ikeda, T and Ishida, T and Nimura-Matsune, K and Funabashi, H and Watanabe, S and Kuroda, A and Hirota, R},
title = {Role of the Pho regulon and genetic reconstruction of a phosphite-dependent Escherichia coli.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {3},
pages = {117-122},
doi = {10.1016/j.jbiosc.2025.05.012},
pmid = {40527655},
issn = {1347-4421},
mesh = {*Regulon/genetics ; *Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Phosphites/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Bacterial Proteins/genetics/metabolism ; },
abstract = {A phosphite (Pt)-dependent biological containment strategy, achieved by introducing a Pt-metabolic pathway and disrupting endogenous phosphate transporters, renders Escherichia coli growth strictly dependent on Pt, a compound rarely detected in natural environments, thereby preventing unintended environmental spread. In this study, we demonstrated that expression of phosphate regulon (Pho regulon) genes was markedly upregulated in a Pt-dependent E. coli strain due to the elimination of phoU, a negative regulator of the Pho regulon, along with the high-affinity phosphate transporter pstSCAB. However, further genetic modification of this strain for detailed analysis was hindered by the presence of multiple antibiotic resistance markers. To overcome this limitation, we reconstructed a Pt-dependent E. coli strain using CRISPR-Cas12a-mediated genome editing, enabling the removal of the antibiotic resistance markers and facilitating subsequent genetic manipulation. Using this strain, we disrupted the PhoBR two-component regulatory genes and found that deletion of phoBR alleviated the constitutive overexpression of Pho regulon genes and partially restored growth of the Pt-dependent strain. These findings provide mechanistic insights and technical advances for the refinement and practical application of Pt-dependent biocontainment strategy.},
}

*Regulon/genetics
*Escherichia coli/genetics/metabolism
*Escherichia coli Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
*Phosphites/metabolism
CRISPR-Cas Systems
Gene Editing
Bacterial Proteins/genetics/metabolism

@article {pmid40514427,
year = {2025},
author = {Liu, L and Chen, S and Lei, Y and Lin, Z and Zhou, R and Zeng, G and Zheng, Z and Liu, W and Zhou, Q and Chen, L},
title = {REPS2 attenuates cancer stemness through inhibiting Wnt signaling by autophagy mediated degradation of β-catenin.},
journal = {Oncogene},
volume = {44},
number = {33},
pages = {2942-2955},
pmid = {40514427},
issn = {1476-5594},
mesh = {Humans ; *Autophagy/genetics ; *beta Catenin/metabolism/genetics ; *Wnt Signaling Pathway/genetics ; *Neoplastic Stem Cells/metabolism/pathology ; Animals ; Mice ; *Lung Neoplasms/pathology/genetics/metabolism ; Cell Line, Tumor ; Proteolysis ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Tumor suppressor genes (TSGs) that regulate the stemness of lung cancer cells remain to be determined. We conducted a genome-wide CRISPR/Cas9-mediated screening and identified REPS2 as a potent TSG that negatively regulates the stemness of lung cancer cells. Its tumor suppressive function was confirmed both in vitro and in vivo. Mechanistically, P62 interacts simultaneously with both β-catenin and REPS2, leading to autophagy-lysosome-mediated degradation of β-catenin and attenuation of Wnt signaling. A β-catenin inhibitor synergizes with inhibitors for driver mutants to induce immunogenic cell death, which could be exploited for enhancing efficacy of tumor immunotherapy.},
}

Humans
*Autophagy/genetics
*beta Catenin/metabolism/genetics
*Wnt Signaling Pathway/genetics
*Neoplastic Stem Cells/metabolism/pathology
Animals
Mice
*Lung Neoplasms/pathology/genetics/metabolism
Cell Line, Tumor
Proteolysis
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid40738806,
year = {2025},
author = {Patel, SJ and Chen, ZJ},
title = {VDAC2 brake release: unleashing inflammation via IFNγ.},
journal = {Trends in pharmacological sciences},
volume = {46},
number = {8},
pages = {695-696},
doi = {10.1016/j.tips.2025.07.001},
pmid = {40738806},
issn = {1873-3735},
mesh = {Humans ; *Interferon-gamma/metabolism ; *Voltage-Dependent Anion Channel 2/metabolism/genetics/antagonists & inhibitors ; *Inflammation/metabolism/drug therapy ; Animals ; *Neoplasms/drug therapy/metabolism/genetics/immunology ; CRISPR-Cas Systems ; DNA, Mitochondrial/metabolism ; },
abstract = {Identification of therapeutic vulnerabilities in cancer remains a high priority for cancer research. A recent CRISPR/Cas9 screen identified that VDAC2 deletion in tumors enhanced their sensitivity to interferon-γ (IFNγ) through the release of mitochondrial DNA (mtDNA) and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. These data suggest that VDAC2 inhibition could enhance antitumor therapies.},
}

Humans
*Interferon-gamma/metabolism
*Voltage-Dependent Anion Channel 2/metabolism/genetics/antagonists & inhibitors
*Inflammation/metabolism/drug therapy
Animals
*Neoplasms/drug therapy/metabolism/genetics/immunology
CRISPR-Cas Systems
DNA, Mitochondrial/metabolism

@article {pmid40695277,
year = {2025},
author = {Sousa, AA and Terrey, M and Sakai, HA and Simmons, CQ and Arystarkhova, E and Morsci, NS and Anderson, LC and Xie, J and Suri-Payer, F and Laux, LC and Roze, E and Forlani, S and Gao, G and Frost, S and Frost, N and Sweadner, KJ and George, AL and Lutz, CM and Liu, DR},
title = {In vivo prime editing rescues alternating hemiplegia of childhood in mice.},
journal = {Cell},
volume = {188},
number = {16},
pages = {4275-4294.e23},
doi = {10.1016/j.cell.2025.06.038},
pmid = {40695277},
issn = {1097-4172},
mesh = {Animals ; *Sodium-Potassium-Exchanging ATPase/genetics/metabolism ; *Hemiplegia/genetics/therapy ; Humans ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; Mutation ; Male ; CRISPR-Cas Systems ; Female ; Genetic Therapy/methods ; Mice, Inbred C57BL ; HEK293 Cells ; Brain/metabolism ; },
abstract = {Alternating hemiplegia of childhood (AHC) is a neurodevelopmental disorder with no disease-modifying treatment. Mutations in ATP1A3, encoding an Na[+]/K[+] ATPase subunit, cause 70% of AHC cases. Here, we present prime editing (PE) and base editing (BE) strategies to correct ATP1A3 and Atp1a3 mutations in human cells and in two AHC mouse models. We used PE and BE to correct five prevalent ATP1A3 mutations with 43%-90% efficiency. AAV9-mediated in vivo PE corrects Atp1a3 D801N and E815K in the CNS of two AHC mouse models, yielding up to 48% DNA correction and 73% mRNA correction in bulk brain cortex. In vivo PE rescued clinically relevant phenotypes, including restoration of ATPase activity; amelioration of paroxysmal spells, motor defects, and cognition deficits; and dramatic extension of animal lifespan. This work suggests a potential one-time PE treatment for AHC and establishes the ability of PE to rescue a neurological disease in animals.},
}

Animals
*Sodium-Potassium-Exchanging ATPase/genetics/metabolism
*Hemiplegia/genetics/therapy
Humans
Mice
*Gene Editing/methods
Disease Models, Animal
Mutation
Male
CRISPR-Cas Systems
Female
Genetic Therapy/methods
Mice, Inbred C57BL
HEK293 Cells
Brain/metabolism

@article {pmid40779454,
year = {2025},
author = {Jain, PK and Mahanty, S and Chittora, H and Henriot, V and Janke, C and Sirajuddin, M and Dhandapany, PS},
title = {Tubulin tyrosine ligase variant perturbs microtubule tyrosination, causing hypertrophy in patient-specific and CRISPR gene-edited iPSC-cardiomyocytes.},
journal = {JCI insight},
volume = {10},
number = {15},
pages = {},
doi = {10.1172/jci.insight.187942},
pmid = {40779454},
issn = {2379-3708},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism ; Gene Editing ; *Cardiomyopathy, Hypertrophic/genetics/pathology ; *Microtubules/metabolism ; Mutation, Missense ; Tubulin/metabolism ; *Peptide Synthases/genetics/metabolism ; Male ; CRISPR-Cas Systems ; Female ; },
abstract = {Hypertrophic cardiomyopathy (HCM) is a hereditary heart condition characterized by either preserved or reduced ejection fraction without any underlying secondary causes. The primary cause of HCM is sarcomeric gene mutations, which account for only 40%-50% of the total cases. Here, we identified a pathogenic missense variant in tubulin tyrosine ligase (TTL p.G219S) in a patient with HCM. We used clinical, genetics, computational, and protein biochemistry approaches, as well as patient-specific and CRISPR gene-edited induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), to demonstrate that the TTL pathogenic variant results in a reduced enzymatic activity and the accumulation of detyrosinated tubulin leading to the disruption of redox signaling, ultimately leading to HCM. Our findings highlight - for the first time to our knowledge - the crucial roles of the TTL variant in cardiac remodeling resulting in disease.},
}

Humans
*Myocytes, Cardiac/metabolism/pathology
Induced Pluripotent Stem Cells/metabolism
Gene Editing
*Cardiomyopathy, Hypertrophic/genetics/pathology
*Microtubules/metabolism
Mutation, Missense
Tubulin/metabolism
*Peptide Synthases/genetics/metabolism
Male
CRISPR-Cas Systems
Female

@article {pmid40778611,
year = {2025},
author = {Brara, Z and Madani, K and Costa, J and Taouzinet, L and Djaoudene, O and Amrane-Abider, M and Bougherra, H and Mouhoubi, K and Brahimi, N and Bouiche, C and Meghlaoui, Z and Mafra, I},
title = {Current Progress on the Detection of Genetically Modified Organisms in Food: From Transgenic Towards Genome-Edited Crops.},
journal = {Comprehensive reviews in food science and food safety},
volume = {24},
number = {5},
pages = {e70243},
doi = {10.1111/1541-4337.70243},
pmid = {40778611},
issn = {1541-4337},
support = {2021.03583.CEECIND/CP1662/CT0012//Fundação para a Ciência e a Tecnologia/ ; UIDB/50006/2020//Fundação para a Ciência e a Tecnologia/ ; 2021.03670.CEECIND/CP1662/CT0011//Fundação para a Ciência e a Tecnologia/ ; DGRSDT/MESRS-Algeria//General Directorate of Scientific Research and Technological Developmen/ ; },
mesh = {*Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; Gene Editing ; *Food, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Progresses in biotechnology, particularly the introduction of genetically modified organisms (GMO) and, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated genome editing, have revolutionized agriculture, prompting the need for robust food-labeling regulations. To meet the GMO legislation requirements, analytical methods for the reliable detection of GMO in food, based either on DNA or protein markers, have been constantly proposed. DNA-based methods relying on polymerase chain reaction (PCR) technologies are the most popular for this purpose, with real-time PCR being the gold standard for GMO quantification. Lately, digital PCR has proved to be a suitable alternative to real-time PCR. The development of rapid, low-cost, user-friendly, and field-deployable tools to quickly generate data on the presence of GMO is crucial, especially with the rapid increase in their complexity and the number of events entering the food market. In this context, alternative technologies based on isothermal amplification and genosensors have emerged. The immunochemical assays in the formats of microtiter plates, lateral flow devices, and immunosensors still occupy a relevant role in GMO detection. Finally, next generation sequencing technologies stand up as tools to face the challenges of detecting unauthorized GMO and genome-edited crops. This review intends to provide a comprehensive overview on the methodologies available for the detection, identification, and quantification of GMO, including gene-edited mutants in foods, while discussing their advantages and limitations, with focus on the latest advances.},
}

*Plants, Genetically Modified/genetics
*Crops, Agricultural/genetics
Gene Editing
*Food, Genetically Modified
CRISPR-Cas Systems

@article {pmid40777742,
year = {2025},
author = {Kalter, N and Fuster-García, C and Silva, A and Ronco-Díaz, V and Roncelli, S and Turchiano, G and Gorodkin, J and Cathomen, T and Benabdellah, K and Lee, C and Hendel, A},
title = {Off-target effects in CRISPR-Cas genome editing for human therapeutics: Progress and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102636},
pmid = {40777742},
issn = {2162-2531},
abstract = {Targeted nucleases, primarily CRISPR-Cas-based systems, have revolutionized genome editing by enabling precise modification of target genes or transcripts. Many pre-clinical and clinical studies leverage this technology to develop treatments for human diseases; however, substantial off-target genotoxicity concerns delay its clinical translation. Despite the development of a wide array of tools, assays, and technologies aimed at identifying and quantifying off-target effects, the absence of standardized guidelines leads to inconsistent practices across studies. This review highlights the key challenges and potential solutions in ensuring the safety of gene editing studies for therapeutic applications, focusing on gRNA design, off-target sites prediction, and off-target activity measurement.},
}

@article {pmid40777740,
year = {2025},
author = {Park, SJ and Lee, GE and Cho, SM and Choi, EH},
title = {Recent applications, future perspectives, and limitations of the CRISPR-Cas system.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {3},
pages = {102634},
pmid = {40777740},
issn = {2162-2531},
abstract = {The CRISPR-Cas system has transformed our ability to edit and modify genomes in eukaryotic cells, offering unmatched precision and broad applicability. By utilizing a programmable RNA protein complex to introduce targeted double-strand breaks, the CRISPR-Cas system enables the correction of pathogenic mutations and the modulation of gene function with unprecedented efficiency. Its broad applicability spans the correction of inherited genetic defects through homology-directed repair to the disruption of deleterious alleles via non-homologous end joining. In this review, we first outline the molecular architecture and mechanistic basis of CRISPR-Cas9 and then consider its latest applications in modeling, drug screening, small-molecule-mediated editing, and treating hereditary, autoimmune, and oncological diseases. Emphasis is placed on the generation of disease-relevant cellular and animal models and on the potential of CRISPR-Cas9-mediated gene therapy to address hitherto intractable disorders. Finally, we discuss current challenges including off-target activity, gene editing efficiency, delivery constraints, and immunogenicity and highlight emerging strategies to overcome these hurdles and broaden the clinical impact of CRISPR-Cas systems.},
}

@article {pmid40777476,
year = {2025},
author = {Walker, MWG and Richard, E and Wiegand, T and Wang, J and Yang, Z and Casas-Ciniglio, AA and Hoffmann, FT and Shahnawaz, H and Gaudet, RG and Arpaia, N and Fernández, IS and Sternberg, SH},
title = {Temperate phages enhance host fitness via RNA-guided flagellar remodeling.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.22.666180},
pmid = {40777476},
issn = {2692-8205},
abstract = {Bacterial flagella drive motility and chemotaxis while also playing critical roles in host-pathogen interactions, as their oligomeric subunit, flagellin, is specifically recognized by the mammalian immune system and flagellotropic bacteriophages. We recently discovered a family of phage-encoded, RNA-guided transcription factors known as TldR that regulate flagellin expression. However, the biological significance for this regulation, particularly in the context of host fitness, remained unknown. By focusing on a human clinical Enterobacter isolate that encodes a Flagellin Remodeling prophage (FRφ), here we show that FRφ exploits the combined action of TldR and its flagellin isoform to dramatically alter the flagellar composition and phenotypic properties of its host. This transformation has striking biological consequences, enhancing bacterial motility and mammalian immune evasion, and structural studies by cryo-EM of host- and prophage-encoded filaments reveal distinct architectures underlying these physiological changes. Moreover, we find that FRφ improves colonization in the murine gut, illustrating the beneficial effect of prophage-mediated flagellar remodeling in a host-associated environment. Remarkably, flagellin-regulating TldR homologs emerged multiple times independently, further highlighting the strong selective pressures that drove evolution of RNA-guided flagellin control. Collectively, our results reveal how RNA-guided transcription factors emerged in a parallel evolutionary path to CRISPR-Cas and were co-opted by phages to remodel the flagellar apparatus and enhance host fitness.},
}

@article {pmid40777396,
year = {2025},
author = {Zhou, S and Yang, S and Xu, J and Zhu, G},
title = {Engineering circular guide RNA and CRISPR-Cas13d-encoding mRNA for the RNA editing of Adar1 in triple-negative breast cancer immunotherapy.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.07.22.666181},
pmid = {40777396},
issn = {2692-8205},
abstract = {Clustered regularly interspaced short palindromic repeat Cas endonuclease (CRISPR-Cas) systems, such as RNA-editing CRISPR-Cas13d, are poised to advance the gene therapy of various diseases. However, their clinical development has been challenged by 1) the limited biostability of linear guide RNAs (lgRNAs) susceptible to degradation, 2) the immunogenicity of prokaryotic microorganism-derived Cas proteins in human that restrains their long-term therapeutic efficacy, and 3) off-targeting gene editing caused by the prolonged Cas expression from DNA vectors. Here, we report the development of highly stable circular gRNAs (cgRNAs) and transiently-expressing Cas13d-encoding mRNA for efficient CRISPR-Cas13d editing of target mRNA. We first optimized cgRNA for CRISPR-Cas13d editing of adenosine deaminase acting on RNA type I (Adar1) transcript for the combination immunotherapy of triple negative breast cancer (TNBC). cgRNAs were synthesized by enzymatic ligation of lgRNA precursors. cgRNAs enhanced biostability with comparable Cas13d-binding affinity relative to lgRNA. Next, using ionizable lipid nanoparticles (LNPs), we co-delivered the resulting Adar1 -targeting cgRNA with an mRNA encoding RfxCas13d (mRNA-RfxCas13d), a widely used Cas13d variant, to TNBC cells. As a result, relative to lgRNA, cgRNA significantly enhanced the efficiency of Adar1 knockdown with minimal collateral activity, which sensitized the cancer cells for cytokine-mediated cell apoptosis. In a 4T1 murine TNBC tumor model in syngeneic mice, Adar1 -targeting cgRNA outperformed lgRNA for tumor immunotherapy in combination with immune checkpoint blockade (ICB). Collectively, these results demonstrate the great potential of cgRNA and mRNA-RfxCas13d for RNA-targeted gene editing.},
}

@article {pmid40775081,
year = {2025},
author = {Chandrasekaran, SN and Alix, E and Arevalo, J and Borowa, A and Byrne, PJ and Charles, WG and Chen, ZS and Cimini, BA and Deng, B and Doench, JG and Ewald, JD and Fritchman, B and Fuller, CJ and Gaetz, J and Goodale, A and Haghighi, M and Han, Y and Hanifehlou, Z and Hennig, H and Hernandez, D and Jacob, CB and James, T and Jetka, T and Kalinin, AA and Komalo, B and Kost-Alimova, M and Krawiec, T and Marion, BA and Martin, G and McCarthy, NJ and Miller, L and Monsees, A and Moshkov, N and Muñoz, AF and Ogier, A and Otrocka, M and Rataj, K and Root, DE and Rubbo, F and Scrace, S and Selinger, DW and Senft, RA and Sommer, P and Thibaudeau, A and Trisorus, S and Valiya Veettil, R and Van Trump, WJ and Wang, S and Warchoł, M and Weisbart, E and Weiss, A and Wiest, M and Zaremba, A and Zinovyev, A and Singh, S and Carpenter, AE},
title = {Morphological map of under- and overexpression of genes in human cells.},
journal = {Nature methods},
volume = {22},
number = {8},
pages = {1742-1752},
pmid = {40775081},
issn = {1548-7105},
support = {MIRA R35 GM122547//U.S. Department of Health & Human Services | NIH | Center for Information Technology (Center for Information Technology, National Institutes of Health)/ ; },
mesh = {Humans ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Cell Painting images offer valuable insights into a cell's state and enable many biological applications, but publicly available arrayed datasets only include hundreds of genes perturbed. The JUMP Cell Painting Consortium perturbed roughly 75% of the protein-coding genome in human U-2 OS cells, generating a rich resource of single-cell images and extracted features. These profiles capture the phenotypic impacts of perturbing 15,243 human genes, including overexpressing 12,609 genes (using open reading frames) and knocking out 7,975 genes (using CRISPR-Cas9). Here we mitigated technical artifacts by rigorously evaluating data processing options and validated the dataset's robustness and biological relevance. Analysis of phenotypic profiles revealed previously undiscovered gene clusters and functional relationships, including those associated with mitochondrial function, cancer and neural processes. The JUMP Cell Painting genetic dataset is a valuable resource for exploring gene relationships and uncovering previously unknown functions.},
}

Humans
*Single-Cell Analysis/methods
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40457553,
year = {2025},
author = {Yuan, Y and Zhou, T and Yang, Z and Zhang, Y and Ye, Z and Liu, L and Zheng, L and Yan, Z and Hu, H and Han, M and Westhof, E and Dai, F and Tong, X},
title = {Doublesex gene in Bombyx mori: CRISPR-mediated female-to-male sex reversal through exon skipping.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5917-5927},
doi = {10.1002/ps.8947},
pmid = {40457553},
issn = {1526-4998},
support = {2023YFD1600901//National key research and development program of China/ ; 2023YFF1103801//National key research and development program of China/ ; SWU-XJLJ202309//Fundamental Research Funds for the Central Universities/ ; SWU-XDPY22011//Fundamental Research Funds for the Central Universities/ ; CARS-18-ZJ0102//China Agriculture Research System of MOF and MARA/ ; CARS-18-ZJ0103//China Agriculture Research System of MOF and MARA/ ; cstc2021jcyj-cxtt0005//Natural Science Foundation of Chongqing, China/ ; },
mesh = {Animals ; *Bombyx/genetics/growth & development ; Male ; Female ; *CRISPR-Cas Systems ; *Sex Determination Processes/genetics ; *Insect Proteins/genetics/metabolism ; Exons ; Gene Editing ; DNA-Binding Proteins ; },
abstract = {BACKGROUND: Sex control technology has great potential to improve the efficiency of economic insect utilization and integrated pest management by enabling targeted population suppression or elimination. The silkworm (Bombyx mori), a lepidopteran model, holds immense economic importance attributable to its unique silk production capabilities. Male silkworms favored over females as a result of their higher yield and quality silk. Doublesex (dsx) is a key gene for sex determination in insects. Previously, inactivation of Bmdsx, among other sex determination-related genes, resulted in intersex individuals rather than a complete female-to-male transformation.

RESULTS: We used CRISPR/Cas9 technology to edit the splice sites of Bmdsx at female-specific exons 3 and 4 to induce transformation of Bmdsx[F] to Bmdsx[M]. The edited genome produced only male-specific Bmdsx[M]. The results revealed that these genetic modifications had significant effects on genital development in female individuals. The edited genome led to the emergence of male-specific gonads in females, causing genital developmental defects. However, it is worth noting that males remained unaffected by these genetic alterations. Furthermore, we conducted a comprehensive analysis of dsx gene structure and explored the potential applicability of this method for inducing female-to-male transgenic transformation in other insects.

CONCLUSIONS: This study demonstrates that precision editing of dsx splice sites can enforce unidirectional sex conversion in insects, disrupting female reproductive capacity without affecting males. Overall, these findings provide valuable insights into our understanding of gonadal development and insect population management. © 2025 Society of Chemical Industry.},
}

Animals
*Bombyx/genetics/growth & development
Male
Female
*CRISPR-Cas Systems
*Sex Determination Processes/genetics
*Insect Proteins/genetics/metabolism
Exons
Gene Editing
DNA-Binding Proteins

@article {pmid40457530,
year = {2025},
author = {Guo, D and Zhao, SY and Su, SC and Bu, LA and Sun, H and Gao, CF and Wu, SF},
title = {Doublesex regulates the development of sex-specific organs in rice stem borer, Chilo suppressalis.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5876-5889},
doi = {10.1002/ps.8941},
pmid = {40457530},
issn = {1526-4998},
support = {//National Key Research and Development Program of China/ ; //National Natural Science Foundation of China/ ; //Fundamental Research Funds for the Central Universities/ ; },
mesh = {Animals ; *Moths/genetics/growth & development ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Phylogeny ; *Sex Determination Processes ; Oryza ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The doublesex (dsx) gene is a crucial component of the insect sex determination pathway, playing a pivotal role in sexual differentiation and development. While the functions of dsx have been extensively characterized in several insect species, its molecular mechanisms and specific roles in sex determination remain largely unexplored in the lepidopteran rice stem borer, Chilo suppressalis (Walker), a significant agricultural pest.

RESULTS: In this study, we identified two male-specific and five female-specific Csdsx transcripts in C. suppressalis. Phylogenetic analyses and multiple sequence alignments revealed that Csdsx is highly conserved among lepidopterans. To investigate its functional roles, we employed CRISPR/Cas9-mediated mutagenesis to generate C. suppressalis mutants. Specifically, target sites were designed on exons 1, 3, 4, and 5 to disrupt the common region (exon 1), female-specific exons (exon 3 and exon 4), and male-specific regions of Csdsx (exon 5). As anticipated, mutants exhibited abnormal development of both external and internal genitalia during the pupal and adult stages. Furthermore, mutations induced sex-specific sterility. Notably, knockout of sex-specific Csdsx isoforms also resulted in abnormal wing patterns and antennal morphology.

CONCLUSION: This study demonstrates the conserved role of Csdsx in sexual differentiation in C. suppressalis and highlights its potential as a molecular target for environmentally friendly pest management strategies against this agricultural pest. © 2025 Society of Chemical Industry.},
}

Animals
*Moths/genetics/growth & development
Female
Male
*Insect Proteins/genetics/metabolism
Phylogeny
*Sex Determination Processes
Oryza
CRISPR-Cas Systems

@article {pmid40417776,
year = {2025},
author = {Lin, M and Zhou, Z and Li, Z and Feng, W and Yang, C and Zhu, Z and Chen, Q},
title = {A dual-probe one-pot RPA-CRISPR/Cas12a: a highly sensitive and rapid method for detection of Phytophthora vignae.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5784-5795},
doi = {10.1002/ps.8932},
pmid = {40417776},
issn = {1526-4998},
support = {2024YFD1400100//National Key R&D Program of China/ ; ZDYF2024XDNY250//Science and Technology special fund of Hainan Province/ ; ZDYF2022XDNY241//Science and Technology special fund of Hainan Province/ ; KYQD(ZR)-20080//Scientific Research Foundation of Hainan University/ ; },
mesh = {*Phytophthora/isolation & purification/genetics ; *Plant Diseases/microbiology/parasitology ; *Vigna/microbiology/parasitology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Phytophthora vignae is the primary pathogen affecting cowpea plants. Early on-site and accurate detection of the causal pathogen is critical for successful management. Therefore, it is both essential and urgent to create a rapid, sensitive, and accurate detection for P. vignae in cowpeas.

RESULTS: In this study, the one-pot RPA-CRISPR/Cas12a assay demonstrated the ability to effectively differentiate closely related oomycete species, exhibiting excellent specificity. The entire detection process can be completed within 15-20 min, with results being visually determined using a lateral flow strip (LFS) or by fluorescence detection under blue or ultraviolet (UV) light. Additionally, we have added dual probes to the one-pot RPA-CRISPR/Cas12a assay detection system, this modification improved reaction efficiency and significantly enhanced sensitivity (200 fg μL[-1]).

CONCLUSION: This study developed a dual-probe one-pot RPA-CRISPR/Cas12a detection platform that demonstrates rapid and highly sensitive identification of P. vignae. The proposed technology enables field-deployable detection without requiring specialized technical expertise, representing a promising approach with substantial potential for practical applications. © 2025 Society of Chemical Industry.},
}

*Phytophthora/isolation & purification/genetics
*Plant Diseases/microbiology/parasitology
*Vigna/microbiology/parasitology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods

@article {pmid40415729,
year = {2025},
author = {Ban, Y and Zhang, Z and Wei, J and Xu, G and Ma, Y and Yin, S and Dong, L and Feng, Z},
title = {Establishment of a CRISPR-based system for rapidly detecting the target-site resistance of American sloughgrass (Beckmannia syzigachne) to Pinoxaden.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5649-5658},
doi = {10.1002/ps.8919},
pmid = {40415729},
issn = {1526-4998},
support = {2023YFD1400501//National Key R&D Program of China/ ; 32372567//National Natural Science Foundation of China/ ; KYCX24-0982//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Herbicide Resistance/genetics ; *Herbicides/pharmacology ; *CRISPR-Cas Systems ; *Poaceae/genetics/drug effects ; *Oxazines/pharmacology ; *Plant Weeds/genetics/drug effects ; Acetyl-CoA Carboxylase/genetics/antagonists & inhibitors ; Mutation ; Plant Proteins/genetics ; Weed Control ; Heterocyclic Compounds, 2-Ring ; },
abstract = {BACKGROUND: Weeds resistant to herbicides pose significant challenges in crop production, making early resistance monitoring crucial for timely control of resistant weeds. Prolonged use of ACCase-inhibiting herbicides, like pinoxaden, has led to the evolution of high-level resistance in weed populations over time. American sloughgrass (Beckmannia syzigachne), is a noxious grass weed, that severely impacts the yield and quality of wheat and rapeseed crops.

RESULTS: To accurately and rapidly detect the mutations in the target gene of B. syzigachne, we developed a novel rapid detection method based on the CRISPR-Cas12b/sgRNA system to evaluate the target mutation at amino acid position 1781 of the ACCase gene. By optimizing various reaction conditions, the novel detection system can assess target-site resistance of B. syzigachne to pinoxaden within 40 min at a constant temperature of 54 °C. This novel system exhibits excellent specificity, high sensitivity, simplicity in procedure, also with time-efficient and high throughput.

CONCLUSION: This study presents an efficient method based on the CRISPR-Cas12b system for rapidly detecting the target-site resistance, which will facilitate the precise management of resistant weeds. © 2025 Society of Chemical Industry.},
}

*Herbicide Resistance/genetics
*Herbicides/pharmacology
*CRISPR-Cas Systems
*Poaceae/genetics/drug effects
*Oxazines/pharmacology
*Plant Weeds/genetics/drug effects
Acetyl-CoA Carboxylase/genetics/antagonists & inhibitors
Mutation
Plant Proteins/genetics
Weed Control
Heterocyclic Compounds, 2-Ring

@article {pmid40396330,
year = {2025},
author = {Yin, H and Ge, Z and Yao, S and Chen, G and Yuan, S and Yang, Q and Deng, W},
title = {Resistance to ACCase-inhibiting herbicides conferred by the Ile-1781-Val mutation in Leptochloa fusca and visual detection of target mutations by RAA-CRISPR/Cas12a.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5581-5588},
doi = {10.1002/ps.8911},
pmid = {40396330},
issn = {1526-4998},
support = {137050535//Qing Lan Project of Yangzhou University/ ; },
mesh = {*Herbicide Resistance/genetics ; *Herbicides/pharmacology ; CRISPR-Cas Systems ; *Acetyl-CoA Carboxylase/antagonists & inhibitors/genetics ; Mutation ; *Poaceae/genetics/drug effects/enzymology ; *Plant Proteins/genetics/antagonists & inhibitors/metabolism ; },
abstract = {BACKGROUND: Leptochloa fusca is a new dominant grass weed in paddy ecosystem of local region in China. The extensive herbicide application has selected the cyhalofop-butyl-resistant L. fusca populations. Here, we aimed to (i) characterize the target-site resistance mechanisms to ACCase inhibitors, and (ii) establish an efficient and rapid method for detecting mutations.

RESULTS: The L. fusca population (SFC-R) was resistant to cyhalofop-butyl (RF = 5.4), metamifop (RF = 5.5), and fenoxaprop-P-ethyl (RF = 6.8). Gene sequencing revealed that the Ile-1781-Val mutation was the molecular basis of resistance. A recombinase-aided amplification (RAA) combined with CRISPR/Cas12a method was successfully developed, which could rapidly and visually detect the single nucleotide variation underling the Ile-1781-Val substitution in L. fusca.

CONCLUSION: This study confirmed that target-site mutation (Ile-1781-Val) endowed resistance to ACCase inhibitors in L. fusca, and represented the first report of applying the RAA-CRISPR/Cas12a assay for mutation detection in resistant weeds. Considering its simplicity and specificity, this technique may be used in monitoring and early warning of resistant weeds. © 2025 Society of Chemical Industry.},
}

*Herbicide Resistance/genetics
*Herbicides/pharmacology
CRISPR-Cas Systems
*Acetyl-CoA Carboxylase/antagonists & inhibitors/genetics
Mutation
*Poaceae/genetics/drug effects/enzymology
*Plant Proteins/genetics/antagonists & inhibitors/metabolism

@article {pmid40304168,
year = {2025},
author = {Xie, QP and Wang, BY and Dou, W and Smagghe, G and Zhang, Q and Wang, JJ},
title = {CRISPR/Cas9-mediated vitellogenin receptor knockout impairs vitellogenin uptake and reproduction in Bactrocera dorsalis.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5043-5051},
doi = {10.1002/ps.8857},
pmid = {40304168},
issn = {1526-4998},
support = {32302340//National Natural Science Foundation of China/ ; 32072422//National Natural Science Foundation of China/ ; SWU-KQ23010//Fundamental Research Funds for the Central Universities/ ; CARS-26//China Agricultural Research System/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Tephritidae/genetics/physiology/metabolism ; *Vitellogenins/metabolism ; *Insect Proteins/genetics/metabolism ; *Egg Proteins/genetics/metabolism ; Reproduction/genetics ; *Receptors, Cell Surface/genetics/metabolism ; Ovary/growth & development/metabolism ; Gene Knockout Techniques ; Male ; },
abstract = {BACKGROUND: Reproduction is a critical process in the insect life cycle, with the ovary serving as the central organ responsible for population maintenance. Successful development of the ovary is dependent on vitellogenin (Vg) transport into oocytes via the vitellogenin receptor (VgR). Exploring the VgR function is crucial for understanding the physiological mechanisms of insect ovarian development. However, the functional role of VgR in Bactrocera dorsalis (Hendel), a notorious agricultural invasive pest with exceptional reproductive plasticity, remains unclear.

RESULTS: Here, we identified BdVgR, an ovary-specific receptor with 1903 amino acids, as a critical determinant of reproductive success. CRISPR/Cas9-mediated BdVgR knockout resulted in a 211-bp genomic deletion spanning exonic (126 bp) and intronic (85 bp) regions, leading to near-complete loss of VgR expression in female adults. Functional analyses revealed that BdVgR deficiency disrupted ovarian Vg (Vg1/Vg2/Vg3) accumulation, impaired ovary maturation, and thus caused severe reproductive defects, including a decrease in the size of the ovaries by 49%, mating rates by 45%, egg production by 38%, and hatching rate by 22%.

CONCLUSION: Collectively, these findings indicate that BdVgR plays a key role in the reproductive process in B. dorsalis, and that disrupting VgR function can inhibit egg production, leading to sterility, which highlights the potential that targeting VgR via CRISPR can create genetically sterile females. Data are discussed with regard to integration of a sterile insect technique approach in the design of novel, efficient and safe pest management tactics. © 2025 Society of Chemical Industry.},
}

Animals
*CRISPR-Cas Systems
Female
*Tephritidae/genetics/physiology/metabolism
*Vitellogenins/metabolism
*Insect Proteins/genetics/metabolism
*Egg Proteins/genetics/metabolism
Reproduction/genetics
*Receptors, Cell Surface/genetics/metabolism
Ovary/growth & development/metabolism
Gene Knockout Techniques
Male

@article {pmid40256962,
year = {2025},
author = {Niu, RC and Zeng, QH and Wang, WJ and Wang, YJ and Liu, TX and Zhang, SZ},
title = {Knockout of silk fibroin genes in Plutella xylostella results in decreased fitness and performance.},
journal = {Pest management science},
volume = {81},
number = {9},
pages = {5010-5020},
doi = {10.1002/ps.8852},
pmid = {40256962},
issn = {1526-4998},
support = {//National Natural Science Foundation of China/ ; //Key R&D Program Project of Ningxia Hui Autonomous Region/ ; },
mesh = {Animals ; *Fibroins/genetics/metabolism ; *Moths/genetics/growth & development/physiology ; Gene Knockout Techniques ; Pupa/growth & development/genetics ; Larva/growth & development/genetics/physiology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Silk/genetics ; *Genetic Fitness ; },
abstract = {BACKGROUND: The ability of insects to spin silk is crucial for their survival, reproduction, and interactions with the environment. The diamondback moth (DBM), Plutella xylostella, a serious agricultural pest, relies on silk production, which influences its behavior and population dynamics.

RESULTS: This study employed CRISPR/Cas9 technology to knock out three genes, that is, silk fibroin heavy chain (PxFibH), silk fibroin light chain (PxFibL), and fibrohexamerin (PxP25), to investigate their roles in silk gland development and related biological traits. We successfully generated PxFibH, PxFibL, and PxP25 knockout mutants, which displayed defective cocoon formation and developed into naked pupae. Further analysis revealed significant alterations in silk gland structure and various biological parameters, including increased larval mortality, prolonged developmental time, reduced pupal weight, and shortened adult lifespan.

CONCLUSIONS: These findings highlight the importance of silk fibroin genes in silk production and growth development in P. xylostella, positioning them as potential targets for innovative pest control strategies. © 2025 Society of Chemical Industry.},
}

Animals
*Fibroins/genetics/metabolism
*Moths/genetics/growth & development/physiology
Gene Knockout Techniques
Pupa/growth & development/genetics
Larva/growth & development/genetics/physiology
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Silk/genetics
*Genetic Fitness

@article {pmid40777797,
year = {2025},
author = {Zhao, F and Chen, F and Yu, H and Fan, S and Bai, M and Xue, J and Zhao, Y and Zuo, X and Fan, C and Zhao, Y},
title = {CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.},
journal = {Fundamental research},
volume = {5},
number = {4},
pages = {1481-1487},
pmid = {40777797},
issn = {2667-3258},
abstract = {Plasmid mutagenesis is an essential step to engineer protein variants with desired properties. Direct generation of mutations on plasmids is a promising method compared to PCR-based methods and subcloning, yet it suffers from the sequence restriction in the generation of single-stranded circular plasmids. Here we demonstrate the CRISPR/Cas system-guided plasmid mutagenesis, a method using gRNA/Cas9 nickase complex to generate single-stranded circular plasmids as polymerization templates for mutagenesis. This method can directly create user-defined mutation libraries on the plasmid. It offers broad sequence programmability and even covers methylated plasmids. We further combine this method with rational design to engineer genome-editing protein FnCpf1. The FnCpf1 variants with mutations in the PAM-binding groove were generated. Several variants expanded the PAM range and exhibited lower off-target effect, which can loosen PAM constraint and enhance target specificity for genome editing. This work presents an effective tool for plasmid mutagenesis and protein engineering studies.},
}

@article {pmid40774991,
year = {2025},
author = {Tabashiri, R and Mahmoodian, S and Pakdel, MH and Shariati, V and Meimandipour, A and Zamani, J},
title = {Comprehensive in vitro and whole-genome characterization of probiotic properties in Pediococcus acidilactici P10 isolated from Iranian broiler chicken.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28953},
pmid = {40774991},
issn = {2045-2322},
mesh = {Animals ; *Pediococcus acidilactici/genetics/isolation & purification/physiology ; *Probiotics ; *Chickens/microbiology ; *Genome, Bacterial ; Whole Genome Sequencing ; Bacterial Adhesion/genetics ; Bacteriocins/genetics ; },
abstract = {This study presents a comprehensive characterization of Pediococcus acidilactici strain P10, a novel probiotic isolated from native broiler chickens, integrating in vitro analyses with whole-genome sequencing. P10 demonstrates promising probiotic attributes, supported by both phenotypic and genomic evidence. The strain was non-hemolytic and exhibited high survival rates under simulated gastrointestinal conditions (95-99% in acidic pH, 55% in bile salts), with genomic analysis confirming the presence of associated stress resistance genes. Importantly, P10 displayed potent broad-spectrum antimicrobial activity against key pathogens, underpinned by the identification of multiple putative bacteriocin-encoding genes. Furthermore, the strain showed strong adherence to intestinal epithelial cells, with corresponding adhesion genes identified in its genome. Beyond these phenotypic-genotypic correlations, P10's whole-genome sequencing revealed significant novel findings. The 1.84 Mb genome confirmed P10 as P. acidilactici and, most notably, identified a complete, functional Type II-A CRISPR-Cas system. This system, with 17 phage-matching spacers, represents a robust antiviral defense mechanism, a key and distinct feature for probiotic application. Additionally, pan-genomic analysis highlighted 59 genes unique to P10 not found in other P. acidilactici strains, suggesting novel metabolic and adaptive capabilities previously uncharacterized within the species. In summary, Pediococcus acidilactici strain P10 is a highly promising probiotic, combining confirmed resilience and antimicrobial action with unique genomic advantages such as its specialized CRISPR-Cas system and novel genetic elements, making it a valuable candidate for applications in animal health and functional foods.},
}

Animals
*Pediococcus acidilactici/genetics/isolation & purification/physiology
*Probiotics
*Chickens/microbiology
*Genome, Bacterial
Whole Genome Sequencing
Bacterial Adhesion/genetics
Bacteriocins/genetics

@article {pmid40774482,
year = {2025},
author = {Geerthana, S and Suresh, K and Prabhakar, N and Rani, BU and Thangaraj, K and Paramasivam, M and Kumar, A and Asokan, R and Karuppannasamy, A},
title = {Harnessing CRISPR/Cas-mediated genetic innovations in lepidopteran insects.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {146574},
doi = {10.1016/j.ijbiomac.2025.146574},
pmid = {40774482},
issn = {1879-0003},
abstract = {CRISPR/Cas-mediated genome editing of eukaryotic organisms gained immense popularity after the monumental publications made in 2012 and 2013 in heterologous editing of Escherichia coli and human kidney cells, respectively. In this regard, CRISPR/Cas has several important applications in agriculture, including gene-function validation and pest management. Recently researchers have contemplated employing this approach in achieving a chemical-free insect pest management strategy called 'genetic pest management'. In this approach both sex ratio and male sterility are achieved in one go by editing precisely the sex-determination genes and spermatogenesis-related genes. In vitro target gene validation is the key before taking up the final field application which is accomplished by employing ribonucleoprotein [RNP] complex combining guide RNA [gRNA] targeting specific regions and Cas protein which cuts the DNA at specific site/s followed by the error-prone repair by non-homologous end joining [NHEJ] resulting loss-of-function of the target gene. CRISPR/Cas-based genetic pest management possesses a lot of potential in Lepidoptera an important species order in Insecta and several globally significant pests. Therefore, the present review focuses on pertinent, up-to-date information on genome editing of the functional and phenotypic genes in Lepidoptera.},
}

@article {pmid40774041,
year = {2025},
author = {Long, J and Wu, J and Xi, Y and Zhang, J and Chen, S and Yang, H and Duan, G},
title = {Association between Type IV-A CRISPR/Cas system and plasmid-mediated transmission of carbapenemase genes in Klebsiella pneumoniae.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128297},
doi = {10.1016/j.micres.2025.128297},
pmid = {40774041},
issn = {1618-0623},
abstract = {The global rise of carbapenem-producing K. pneumoniae is largely attributed to plasmid-mediated transmission of carbapenemase genes. Type IV-A CRISPR/Cas system is mainly located on plasmids in K. pneumoniae and involved in plasmid competition. However, the role of Type IV-A system in the dissemination of carbapenemase genes in K. pneumoniae remains unclear. Here, we comprehensively investigated the relationship between Type IV-A system and plasmid-mediated transmission of carbapenemase genes based on 152 K. pneumoniae clinical strains and 46226 K. pneumoniae public genomes available in NCBI database. We found that the presence of Type IV-A system was positively associated with blaNDM-1, blaNDM-5, blaOXA-48, and blaVIM-1 but negatively related to blaKPC-2, blaKPC-3,blaIMP and blaOXA-181. Additionally, plasmids carrying Type IV-A system were predominantly the vehicles of blaNDM-1 gene. Protospacer search revealed that Type IV-A system frequently matched conjugation transfer region of blaKPC-2-related IncF plasmids, especially IncFIB(K)/IncFII(K), IncFII(pHN7A8)/IncR, and IncFIB(pQil)/IncFII(K) plasmids. The prevalence of self-targeting event further highlighted the interference mechanism of transcriptional repression proposed by Type IV system. Despite frequent targeting of IncF plasmids by Type IV-A system, different types of IncF plasmids displayed varying distribution between CRISPR-positive and -negative genomes, thereby suggesting a differentiated response of Type IV-A system to IncF plasmids. Our results underscore complex interactions between Type IV-A system and plasmid-mediated carbapenemase genes, revealing its significant role in shaping the transmission dynamics of carbapenemase-encoding plasmids.},
}

@article {pmid40773238,
year = {2025},
author = {Pai, VJ and Shan, H and Donaldson, CJ and Vaughan, JM and De Souza, EV and O'Connor, C and Liem, M and Pinto, AFM and Diedrich, J and Saghatelian, A},
title = {CRISPR-Cas9 screening reveals microproteins regulating adipocyte proliferation and lipid metabolism.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2506534122},
doi = {10.1073/pnas.2506534122},
pmid = {40773238},
issn = {1091-6490},
support = {F32 DK132927/DK/NIDDK NIH HHS/United States ; R01 GM102491/GM/NIGMS NIH HHS/United States ; RC2 DK129961/DK/NIDDK NIH HHS/United States ; Chair//Ferring Foundation/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Cell Proliferation/genetics ; 3T3-L1 Cells ; *Lipid Metabolism/genetics ; *Adipocytes/metabolism/cytology ; Open Reading Frames/genetics ; Cell Differentiation/genetics ; Micropeptides ; },
abstract = {Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction of mice using ribosome profiling. Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR/Cas9 single guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. The smORFs on the 5'- and 3'-untranslated regions (i.e., upstream smORFs (uORFs) and downstream smORFs (dORFs)) of functional genes can exert activity through cis-regulatory effects of the main ORF on these messenger RNAs (mRNAs), such as uORFs of MDM2 that impact proliferation. However, other smORFs, especially those from mRNAs with no other ORFs, point to a functional microprotein. Indeed, we tested a candidate smORF 1183 from a long noncoding RNA 923011K14Rik and demonstrated that the microprotein regulates adipocyte differentiation. These findings highlight the potential of CRISPR/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.},
}

Animals
Mice
*CRISPR-Cas Systems/genetics
*Cell Proliferation/genetics
3T3-L1 Cells
*Lipid Metabolism/genetics
*Adipocytes/metabolism/cytology
Open Reading Frames/genetics
Cell Differentiation/genetics
Micropeptides

@article {pmid40773236,
year = {2025},
author = {Ogawa, Y and Nguyen, DVM and Ogawa, A and Rasband, MN},
title = {Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2500726122},
doi = {10.1073/pnas.2500726122},
pmid = {40773236},
issn = {1091-6490},
support = {NS122073//HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; N/A//Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF)/ ; },
mesh = {*Gene Editing/methods ; *Synapses/metabolism/genetics ; Animals ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Axons/metabolism ; Mice ; Neurons/metabolism ; Dependovirus/genetics ; Mitochondria/metabolism ; *Carrier Proteins/genetics/metabolism ; },
abstract = {The visualization and manipulation of proteins in neurons is widely used to deduce their functions. While every experimental approach has limitations, the concurrent knock-in and knockout of two different proteins can be especially challenging. To this end, we developed Hide-and-Seek genome editing, which allows the simultaneous visualization and knockout of proteins in neurons using Adeno-associated viral vectors and the CRISPR/Cas9 system. We demonstrate the efficacy and flexibility of this method for rapid, efficient, and simultaneous knock-in and knockout of proteins in vitro and in vivo, at the synapse, axon initial segment (AIS), nucleus, and mitochondria. Using Hide-and-Seek, we show that the scaffolding protein Gephyrin is required for the proper assembly of axo-axonic synapses at the AIS.},
}

*Gene Editing/methods
*Synapses/metabolism/genetics
Animals
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
*Axons/metabolism
Mice
Neurons/metabolism
Dependovirus/genetics
Mitochondria/metabolism
*Carrier Proteins/genetics/metabolism

@article {pmid40773116,
year = {2025},
author = {Patil, C and Priyanka, R and Harshitha, BM and Oshik, S and Yashwanth, S and Darshan, BR and Patil, S and Prajwal, KA and Naik, P and Goudanavar, P and Mallamma, T},
title = {Advanced nanotheranostic approaches for targeted glioblastoma treatment: a synergistic fusion of CRISPR-Cas gene editing, AI-driven tumor profiling, and BBB-modulation.},
journal = {Medical oncology (Northwood, London, England)},
volume = {42},
number = {9},
pages = {413},
pmid = {40773116},
issn = {1559-131X},
mesh = {*Glioblastoma/genetics/therapy/pathology ; Humans ; *Blood-Brain Barrier/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brain Neoplasms/genetics/therapy/pathology ; *Theranostic Nanomedicine/methods ; Animals ; Nanoparticles ; },
abstract = {Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. It is hard to treat because it is very invasive, has a lot of genetic variation, and the blood-brain barrier (BBB) limits its growth. Traditional GBM treatments, including surgery, radiation and chemotherapy have only marginally improved survival requiring a paradigm shift. This review starts a new way of thinking about how to treat GBM by combining multi-responsive nanotheranostics, next-generation genome engineering, and AI-enabled stratification. The study compares smart nanocarriers that can transport payloads accurately and only when needed. CRISPR/Cas systems are useful for directly editing oncogenes, controlling GBM signaling cascades, making resistant cells sensitive again, and changing the ways that cells avoid the immune system. Nanoparticle-mediated CRISPR delivery and BBB disruption approaches, including targeted ultrasound, receptor-mediated transcytosis, and exosome-based delivery, could help get around the neuroprotective shield without damaging it. This review investigates the molecular aspects of the BBB, highlighting its dual role as a metabolic and efflux transporter-mediated barrier, and assesses cutting-edge nanocarrier systems, in particular hybrid exosome-nanoparticle designs, that aim to circumvent these limitations. Additionally, it explores the current limitations and possible future applications of gene editing tools and AI models for navigating the genomic instability and ever-changing tumor microenvironment of GBM. This study also discusses the ethical and legal issues related to these developing technologies, therefore stressing the need for clinically feasible, ethically controlled innovation pipelines. With an emphasis on cutting-edge research, this review delves into emerging areas such as nanotheranostics, cellular composition of GBM, CRISPR-based gene editing, AI-enabled precision medicine, and targeted disruption of the blood-brain barrier.},
}

*Glioblastoma/genetics/therapy/pathology
Humans
*Blood-Brain Barrier/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Brain Neoplasms/genetics/therapy/pathology
*Theranostic Nanomedicine/methods
Animals
Nanoparticles

@article {pmid40772537,
year = {2025},
author = {Yin, Y and Li, Z and Liu, C and Zhang, XB and Ou, Q},
title = {CRISPR-Based Live Cell Biosensing: Innovations in Signal Labeling Strategies and Delivery Systems.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01973},
pmid = {40772537},
issn = {2379-3694},
abstract = {CRISPR technology provides unprecedented precision for molecular imaging and target detection at the cellular level. However, interested researchers inevitably encounter challenges, including weak labeling signals, low delivery efficiency, and off-target effects when establishing application frameworks. Therefore, discussing signal labeling strategies and delivery systems is crucial to further improving the performance of CRISPR-based live cell biosensing. In this review, we first focus on signal labeling strategies for CRISPR-based cellular imaging, including fluorescent protein fusion, fluorescent protein recruitment, RNA modification and hybridization, and irrelevant signal reporters. Depending on the effector proteins and application scenarios, selecting the appropriate signal labeling method can help improve imaging sensitivity and signal intensity. Second, we summarize materials that can be used for CRISPR intracellular delivery, such as nanoparticles, nanosheets, and other nanomaterials. Some nanomaterials have been shown to further enhance the activity of effector proteins, in addition to facilitating the cellular entry of active components. Furthermore, we discuss the challenges and future directions of CRISPR imaging technologies. By integrating multidisciplinary innovations, the CRISPR-based live cell biosensor holds promise as a next-generation visualization tool for life science research and precision medicine.},
}

@article {pmid40771633,
year = {2025},
author = {Sleckman, BP and Chen, BR},
title = {THE GID COMPLEX REGULATES DNA END PROCESSING DURING DNA DOUBLE-STRAND BREAK REPAIR BY NONHOMOLOGOUS END JOINING.},
journal = {Transactions of the American Clinical and Climatological Association},
volume = {135},
number = {},
pages = {281-293},
pmid = {40771633},
issn = {0065-7778},
mesh = {*DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Humans ; Genomic Instability ; CRISPR-Cas Systems ; Animals ; *Ubiquitin-Protein Ligases/metabolism/genetics ; DNA Repair ; },
abstract = {Genome instability is a hallmark of cancer, allowing for clonal evolution and improved tumor fitness. The mis-repair of DNA double-strand breaks (DSBs) is a major source of genome instability. DNA DSBs are normally repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). The nucleolytic resection of broken DNA ends generates single-stranded DNA (ssDNA) overhangs that are required for HR, but inhibitory to NHEJ. DNA end resection must be prevented in nondividing cells where NHEJ is the only active DSB repair pathway. Using a novel whole genome gRNA CRISPR/Cas9 screen, we identified the GID complex as functioning to protect DNA ends from nucleolytic resection. The GID complex contains multiple E3 ubiquitin ligase subunits and regulates the expression and function of pro-resection machinery. Thus, by antagonizing DNA end resection GID may prevent homology-mediated joining leading to aberrant DSB repair and genome instability in normal and cancerous nondividing cells.},
}

*DNA Breaks, Double-Stranded
*DNA End-Joining Repair
Humans
Genomic Instability
CRISPR-Cas Systems
Animals
*Ubiquitin-Protein Ligases/metabolism/genetics
DNA Repair

@article {pmid40771623,
year = {2025},
author = {Sharpe, AH},
title = {THE BIOLOGY BEHIND PD-1 CHECKPOINT BLOCKADE.},
journal = {Transactions of the American Clinical and Climatological Association},
volume = {135},
number = {},
pages = {169-183},
pmid = {40771623},
issn = {0065-7778},
mesh = {Humans ; *Programmed Cell Death 1 Receptor/antagonists & inhibitors/immunology/metabolism/genetics ; *Immune Checkpoint Inhibitors/therapeutic use ; *Neoplasms/immunology/genetics/drug therapy ; CRISPR-Cas Systems ; Animals ; Signal Transduction/drug effects ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Programmed death 1 (PD-1) pathway inhibitors have transformed cancer therapy, leading to durable responses in some patients. However, many patients do not benefit from PD-1 blockade therapy, which highlights the critical need to identify new therapeutic targets to complement PD-1 pathway inhibitors. To address this need, we have developed an in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-based screening platform to discover novel regulators of anti-tumor immunity. In this article, I will first discuss the biology of the PD-1 pathway and its role in regulating anti-tumor immunity. Next, I will introduce our innovative CRISPR-based platforms designed for conducting gene screens in mature immune cell lineages and for enabling gene perturbation without stimulating or manipulating immune cells, two approaches that can affect immune cell development and function. In addition, I will illustrate how these platforms facilitate discovery of new targets that can promote anti-tumor immunity and their potential to lead to more effective cancer therapies.},
}

Humans
*Programmed Cell Death 1 Receptor/antagonists & inhibitors/immunology/metabolism/genetics
*Immune Checkpoint Inhibitors/therapeutic use
*Neoplasms/immunology/genetics/drug therapy
CRISPR-Cas Systems
Animals
Signal Transduction/drug effects
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40770768,
year = {2025},
author = {Wu, L and Zhu, X and Liu, Y and Zhao, D and Yu, BC and Wei, Z and Lin, X and Qi, LS},
title = {Identification of replicative aging and inflammatory aging signatures via whole-genome CRISPRi screens.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {233},
pmid = {40770768},
issn = {1474-760X},
support = {R21AG077193//NIH National Institute of Aging/ ; no. 2046650//the National Science Foundation CAREER award/ ; },
mesh = {Humans ; *Cellular Senescence/genetics ; *Inflammation/genetics ; *Aging/genetics ; Genome-Wide Association Study ; Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Genomics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {BACKGROUND: Aging is a major risk factor for chronic diseases and cancer. Cellular aging, particularly in adult stem cells, offers a high-throughput framework for dissecting the molecular mechanisms of aging.

RESULTS: We perform multiple genome-wide CRISPR interference (CRISPRi) screenings in human primary mesenchymal stem cells derived from adipose tissue during either replicative senescence or inflammation-induced senescence. These screens reveal distinct sets of potential novel regulators specific to each senescence pathway. Combining our perturbation-based functional genomic data with 405 genome-wide association study datasets, including 50 aging-related studies, we find that the inflammatory aging signatures identified from CRISPRi screenings are significantly associated with diverse aging processes, suggesting novel molecular signatures for analyzing and predicting aging status and aging-related disease.

CONCLUSIONS: The signatures verified through comprehensive functional genomics and genetic analyses may provide new targets for modulating the aging process and enhancing the quality of cell therapy products.},
}

Humans
*Cellular Senescence/genetics
*Inflammation/genetics
*Aging/genetics
Genome-Wide Association Study
Mesenchymal Stem Cells/metabolism/cytology
*CRISPR-Cas Systems
Genomics
Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Human

@article {pmid40770375,
year = {2025},
author = {Aldiss, Z and Lam, Y and Robinson, H and Dixon, R and Steinhardt, L and Crisp, P and Godwin, I and Borrell, A and Hickey, L and Massel, K},
title = {The PIN2 ortholog in barley modifies root gravitropism and architecture.},
journal = {The plant genome},
volume = {18},
number = {3},
pages = {e70061},
doi = {10.1002/tpg2.70061},
pmid = {40770375},
issn = {1940-3372},
support = {DP190102185//Australian Research Council/ ; LP200200927//ARC Linkage project/ ; //Australian Government Research Training Program and InterGrain/ ; FT220100350//ARC Future Fellowship/ ; },
mesh = {*Hordeum/genetics/growth & development/physiology ; *Plant Roots/genetics/growth & development/anatomy & histology/physiology ; *Gravitropism/genetics ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Roots provide the critical interface where plants acquire nutrients and water, but our limited understanding of the genetic controls modulating root system architecture (RSA) in crop species constrains opportunities to develop future cultivars with improved root systems. However, there is vast knowledge of root developmental genes in model plant species, which has the potential to accelerate progress in crops with more complex genomes, particularly given that genome editing protocols are now available for most species. PIN-FORMED2 (PIN2) encodes a root-specific polar auxin transporter, where its absence resulted in roots being unable to orient themselves using gravity, producing a significantly wider root system. To explore the role of PIN2 in a cereal crop, we used CRISPR/Cas9 (where CRISPR is clustered regularly interspaced short palindromic repeats) editing to knockout of PIN2 in barley (Hordeum vulgare). Like Arabidopsis, the roots of barley pin2 loss-of-function mutants displayed an agravitropic response at seedling growth stages, resulting in a significantly shallower and wider root system at later growth stages. Notably, despite the significant change in RSA, there was no change in shoot architecture or total shoot biomass, with an insensitivity to the effects of higher planting density. We discuss the future challenges and opportunities to harness the PIN2 pathway to optimize RSA in crops for a range of production scenarios without a shoot trade-off.},
}

*Hordeum/genetics/growth & development/physiology
*Plant Roots/genetics/growth & development/anatomy & histology/physiology
*Gravitropism/genetics
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40770192,
year = {2025},
author = {Erkek, F and Kizilkaya, R and Baybara, S and Gulden, G and Sert, B and Yazicilar, MS and Yilmaz, MC and Odabas, SP and Bal, E and Yelgen, G and Teymur, T and Ay, Y and Dogdu, S and Tiryaki, NN and Baran, B and Gelsin, B and Araz, H and Cavrar, I and Tastan, C},
title = {CRISPR.BOT an autonomous platform for streamlined genetic engineering and molecular biology applications.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28699},
pmid = {40770192},
issn = {2045-2322},
support = {ÜÜBAP-YP-2020-005//Uskudar University Scientific Research Studies/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Engineering/methods ; Green Fluorescent Proteins/genetics ; Synthetic Biology/methods ; HEK293 Cells ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In the realm of synthetic biology, the pursuit of streamlined experimental processes has given rise to innovative technologies poised to redefine research paradigms. Traditional workflows, laden with laborious and error-prone stages, incur prolonged timelines and escalating costs. Autonomous liquid processing systems emerge as promising tools, holding the potential to enhance precision and efficiency. Yet, the adoption of fully automated systems is impeded by prohibitive costs and maintenance expenses. This study introduces the CRISPR.BOT as a transformative solution, a pioneering autonomous genetic engineering platform designed to revolutionize molecular biology practices. The CRISPR.BOT demonstrates its prowess through a series of pivotal experiments, ranging from the efficient transfer of green fluorescent protein (GFP) encoding plasmid DNA into bacterial hosts to intricate manipulations involving lentiviral transduction and CRISPR-Cas9-mediated genetic editing in human cell lines. Notably, the CRISPR.BOT achieves exceptional results in single-cell subcloning, yielding GFP + CRISPR-gRNA + cells with unprecedented purity levels of 90-100%. Moreover, this study underscores the CRISPR.BOT's potential to facilitate safe genetic engineering practices, enabling researchers to work with pathogenic microorganisms like the SARS CoV-2 virus without direct human contact. An exploration into the cost-effectiveness of LEGO Mindstorms robots reveals their suitability for daily laboratory routines, presenting a cost reduction of up to tenfold compared to commercial alternatives. In conclusion, the CRISPR.BOT emerges as a transformative force in synthetic biology, offering a pathway to redefine experimental processes, enhance precision, and unlock new possibilities in genetic engineering. This research marks a paradigm shift where automation and innovation converge, empowering researchers to navigate uncharted territories with confidence and efficiency.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Genetic Engineering/methods
Green Fluorescent Proteins/genetics
Synthetic Biology/methods
HEK293 Cells
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40759748,
year = {2025},
author = {Wolf, B and Shehu, P and Brenker, L and von Bachmann, AL and Kroell, AS and Southern, N and Holderbach, S and Eigenmann, J and Aschenbrenner, S and Mathony, J and Niopek, D},
title = {Rational engineering of allosteric protein switches by in silico prediction of domain insertion sites.},
journal = {Nature methods},
volume = {22},
number = {8},
pages = {1698-1706},
pmid = {40759748},
issn = {1548-7105},
mesh = {*Protein Engineering/methods ; Humans ; Allosteric Regulation ; Machine Learning ; Escherichia coli/genetics/metabolism ; Allosteric Site ; Computer Simulation ; Protein Domains ; CRISPR-Cas Systems ; *Proteins/chemistry/genetics ; },
abstract = {Domain insertion engineering is a powerful approach to juxtapose otherwise separate biological functions, resulting in proteins with new-to-nature activities. A prominent example are switchable protein variants, created by receptor domain insertion into effector proteins. Identifying suitable, allosteric sites for domain insertion, however, typically requires extensive screening and optimization. We present ProDomino, a machine learning pipeline to rationalize domain recombination, trained on a semisynthetic protein sequence dataset derived from naturally occurring intradomain insertion events. ProDomino robustly identifies domain insertion sites in proteins of biotechnological relevance, which we experimentally validated in Escherichia coli and human cells. Finally, we used light- and chemically regulated receptor domains as inserts and demonstrate the rapid, model-guided creation of potent, single-component opto- and chemogenetic protein switches. These include novel CRISPR-Cas9 and -Cas12a variants for inducible genome engineering in human cells. Our work enables one-shot domain insertion engineering and substantially accelerates the design of customized allosteric proteins.},
}

*Protein Engineering/methods
Humans
Allosteric Regulation
Machine Learning
Escherichia coli/genetics/metabolism
Allosteric Site
Computer Simulation
Protein Domains
CRISPR-Cas Systems
*Proteins/chemistry/genetics

@article {pmid40704969,
year = {2025},
author = {Li, J and Lau, CH and Wang, J and Wu, W and Huang, Z and Chen, X and Li, J and Huang, Y and Wang, T and Li, Y and Zhao, Z and Xu, M and Chen, G and Tong, S and Zhu, H},
title = {Rapid, multiplex, one-pot CRISPR/Dx system for detecting cancer fusion genes.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {31},
pages = {6304-6316},
doi = {10.1039/d5ay00783f},
pmid = {40704969},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/diagnosis ; Proto-Oncogene Proteins/genetics ; *Oncogene Proteins, Fusion/genetics ; Protein-Tyrosine Kinases/genetics ; Gene Fusion ; },
abstract = {Targeted therapies directed at fusion genes have proven remarkably effective against cancers. Therefore, the rapid and reliable identification of cancer fusion genes can guide subsequent therapeutic treatment and predict prognosis. By integrating the RT-RPA and CRISPR/Cas12a approaches, we developed a one-pot CRISPR/Dx system for the rapid and multiplex detection of cancer fusion genes. A tube with unique assemblies was created using 3D printing technology to realize this application. As proof of principle, we demonstrated the feasibility of the one-pot CRISPR/Dx system in detecting lung cancer by targeting ROS1 fusions. The performance of the one-pot CRISPR/Dx detection system was comparable to a two-tube-based testing platform. When tested with synthetic RNA fusions, both approaches efficiently detected all 14 ROS1 fusions with an LOD in the range of 5-10 copies per μL, without generating a background signal, even in the presence of a large excess of wild-type RNA. The total reaction time for both approaches was 30 minutes. Notably, the one-pot CRISPR/Dx detection system minimized the operation steps and aerosol contamination without compromising detection sensitivity and specificity. Furthermore, its diagnostic power was validated using clinical samples. Thus, we successfully developed a rapid, multiplex, one-pot CRISPR/Dx detection system for detecting 14 clinically relevant ROS1 fusions with high sensitivity and specificity. It is also cost-effective and simple to operate, thereby realizing the ultimate goal of establishing CRISPR/Dx as the paragon of cancer diagnostics for home self-testing and point-of-care testing.},
}

Humans
*CRISPR-Cas Systems/genetics
*Lung Neoplasms/genetics/diagnosis
Proto-Oncogene Proteins/genetics
*Oncogene Proteins, Fusion/genetics
Protein-Tyrosine Kinases/genetics
Gene Fusion

@article {pmid40695391,
year = {2025},
author = {Bui, VKH and Trung, LG and Do, HH and Tran, NT and Nguyen, MK},
title = {Unlocking the algae toolbox: Cutting-edge tools for environmental and biotechnological solutions.},
journal = {Biotechnology advances},
volume = {83},
number = {},
pages = {108652},
doi = {10.1016/j.biotechadv.2025.108652},
pmid = {40695391},
issn = {1873-1899},
mesh = {*Biotechnology/methods ; *Microalgae/genetics ; CRISPR-Cas Systems ; Biofuels ; Genetic Engineering ; Ecosystem ; },
abstract = {Algae are highly adaptable photosynthetic organisms with growing relevance in biotechnology, environmental management, and sustainable industries. Recent advancements have expanded the "Algae Toolbox", integrating cutting-edge screening, monitoring, characterization, and large-scale production technologies. This current work critically examines the latest developments in algal research, including CRISPR-Cas9 genetic engineering, high-throughput omics, machine learning-driven modeling, and remote sensing-based ecosystem monitoring. Moreover, innovations in optical sensors, flow cytometry, biosensors, and environmental DNA have further improved precision in tracking algal dynamics. These advances support industrial scale-up, boosting productivity and sustainability in biofuels, pharmaceuticals, aquaculture, and bioremediation. However, large-scale production still faces challenges in economic viability and environmental sustainability. Future research must incorporate artificial intelligence, internet of things-enabled sensing, and synthetic biology for optimized cultivation and bioproduct harvesting. This review provides a comprehensive overview of emerging tools and methodologies, emphasizing their role in advancing algal biotechnology and global sustainability.},
}

*Biotechnology/methods
*Microalgae/genetics
CRISPR-Cas Systems
Biofuels
Genetic Engineering
Ecosystem

@article {pmid40663809,
year = {2025},
author = {Wang, J and Tian, Y and Zhang, K and Zhao, Y},
title = {A high-sensitivity ECL biosensor for single-cell analysis: Integrating CRISPR/Cas12a and entropy-driven amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109045},
doi = {10.1016/j.bioelechem.2025.109045},
pmid = {40663809},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; Humans ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; *Entropy ; *Luminescent Measurements/methods ; *Nucleic Acid Amplification Techniques/methods ; *Electrochemical Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study presents a homogeneous electrochemiluminescence (ECL) biosensor for ultrasensitive detection of HEK293 cells by targeting the hERG potassium channel at the single-cell level. The biosensor integrates multiple signal amplification steps, including photocleavable DNA-antibody conjugates, entropy-driven strand displacement, T7 RNA polymerase-mediated transcription, and CRISPR/Cas12a-mediated trans-cleavage. This cascade enables precise and robust signal enhancement. A key feature of the system is its ability to generate a clearly measurable ECL response from as little as a single HEK293 cell, without the need for signal averaging. Specificity was confirmed using unrelated cell lines and mismatched DNA sequences, with minimal background observed in negative controls. Optimization of key parameters-such as enzyme concentrations, reaction times, and duplex composition-ensured consistent and reproducible performance. ECL measurements were conducted under a defined voltage scan (0-1.3 V) in a three-electrode system, and emission signals were recorded using a photomultiplier tube. This biosensor demonstrates the feasibility of single-cell detection with high sensitivity and specificity, offering a promising platform for future applications in cell-based analysis and molecular diagnostics.},
}

*Biosensing Techniques/methods
Humans
*Single-Cell Analysis/methods
*CRISPR-Cas Systems
HEK293 Cells
*Entropy
*Luminescent Measurements/methods
*Nucleic Acid Amplification Techniques/methods
*Electrochemical Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40652737,
year = {2025},
author = {Shi, Z and Yan, Y and Zhu, R and Zhu, X and Hu, K and Yue, Y and Xu, W and Xuan, M and Gan, X and Yang, Z and Zhang, Y and Cao, Z},
title = {DUXA regulates the early development of porcine cloned embryos.},
journal = {Theriogenology},
volume = {247},
number = {},
pages = {117563},
doi = {10.1016/j.theriogenology.2025.117563},
pmid = {40652737},
issn = {1879-3231},
mesh = {Animals ; Swine/embryology/genetics ; *Cloning, Organism/veterinary ; *Embryonic Development/genetics/physiology ; Nuclear Transfer Techniques/veterinary ; *Gene Expression Regulation, Developmental/physiology ; *Homeodomain Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Embryo, Mammalian ; Blastocyst/physiology ; Female ; CRISPR-Cas Systems ; },
abstract = {Abnormal zygotic genome activation (ZGA) during the early development of somatic cell nuclear transfer (SCNT) embryos is one of the main reasons for the low cloning efficiency. The double homeobox (DUX) family, which includes important transcription factors in mammals, has been shown to play an important role in the ZGA process in mice. However, the role of DUXA, a member of the DUX family, in the early development of porcine somatic cloned embryos is unknown. Here, CRISPR/Cas9 gene editing and lentiviral infection technologies were used to construct stable DUXA knockout and overexpression cell lines for the production of SCNT embryos. Compared with that of wild-type (WT) SCNT embryos, the blastocyst rate of DUXA knockout embryos was significantly lower (P < 0.05), whereas the blastocyst rate of DUXA-overexpressing embryos was significantly greater (P < 0.05). Moreover, RT‒qPCR results revealed that DUXA knockout significantly reduced the expression levels of ZGA-related genes (TDG, SNAI1, RSRP1, TFAP2C, ZSCAN4, LEUTX, and KLF17) (P < 0.05). Additionally, in DUXA-overexpressing embryos, the mRNA levels of TDG, SNAI1, RSRP1, and TFAP2C significantly decreased (P < 0.05), whereas the ZSCAN4, LEUTX, and KLF17 mRNA levels increased (P < 0.05). These findings suggest that DUXA regulates the early development of porcine SCNT embryos by modulating the expression of ZGA-related genes. This research provides significant insights into the potential mechanisms of early embryo loss in porcine SCNT.},
}

Animals
Swine/embryology/genetics
*Cloning, Organism/veterinary
*Embryonic Development/genetics/physiology
Nuclear Transfer Techniques/veterinary
*Gene Expression Regulation, Developmental/physiology
*Homeodomain Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Embryo, Mammalian
Blastocyst/physiology
Female
CRISPR-Cas Systems

@article {pmid40651075,
year = {2025},
author = {Xiao, X and Lu, Y and Zhang, J and Ni, W and Liu, J and Li, C and Yao, Q and Sun, Y and Zhang, GJ and Zhang, Y and Qin, Y and Zhou, Y},
title = {Rolling circle amplification and CRISPR/Cas14a with nanozyme for electrochemical detecting miRNA-205 in NPC-derived exosomes.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109046},
doi = {10.1016/j.bioelechem.2025.109046},
pmid = {40651075},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Electrochemical Techniques/methods ; *Exosomes/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/genetics/diagnosis ; Limit of Detection ; *Nasopharyngeal Neoplasms/genetics/diagnosis ; Nanowires/chemistry ; Platinum/chemistry ; },
abstract = {Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, and early detection of biomarkers like miRNA-205 (miR-205) is critical for improving prognosis. However, accurate detection of miR-205 remains challenging due to its low abundance and matrix interference. Herein, an ultrasensitive electrochemical biosensor integrating Pt nanowires/MXene (PtNWs/MXene), rolling circle amplification (RCA), and CRISPR/Cas14a was developed for detecting exosomal miR-205. The dual characteristics of PtNWs/MXene (differential adsorption capacity for intact and cleaved DNA; HRP-like nanozyme activity) enable the conversion of the miR-205-triggered RCA-Cas14a cascade reaction into significant electrochemical signal changes. This biosensor eliminates the requirement for signal probe labeling of the electrode-modified DNA. Moreover, the enzyme-mimicking catalytic activity of PtNWs/MXene enables the catalysis of numerous 3,3',5,5'-tetramethylbenzidine (TMB) molecules, realizing a "one-to-many" signal amplification effect that significantly improves detection sensitivity. The biosensor achieves a detection limit of 4.6 aM (50 aM-10 pM linear range) and distinguishes single-base mismatches. Clinical validation confirmed its ability to differentiate NPC patients from healthy individuals, aligning with qRT-PCR results. By adjusting the RCA template, this strategy can be adapted for diverse RNA/DNA targets, offering a versatile platform for early disease diagnosis.},
}

*MicroRNAs/analysis/genetics
Humans
*Electrochemical Techniques/methods
*Exosomes/chemistry/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Nasopharyngeal Carcinoma/genetics/diagnosis
Limit of Detection
*Nasopharyngeal Neoplasms/genetics/diagnosis
Nanowires/chemistry
Platinum/chemistry

@article {pmid40604285,
year = {2025},
author = {Chen, AXY and Yap, KM and Kim, JS and Sek, K and Huang, YK and Dunbar, PA and Wiebking, V and Armitage, JD and Munoz, I and Todd, KL and Derrick, EB and Nguyen, D and Tong, J and Chan, CW and Hoang, TX and Audsley, KM and van Elsas, MJ and Middelburg, J and Lee, JN and de Menezes, MN and Cole, TJ and Li, J and Scheffler, C and Scott, AM and Mackay, LK and Waithman, J and Oliaro, J and Harrison, SJ and Parish, IA and Lai, J and Porteus, MH and House, IG and Darcy, PK and Beavis, PA},
title = {Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery.},
journal = {Nature},
volume = {644},
number = {8075},
pages = {241-251},
pmid = {40604285},
issn = {1476-4687},
mesh = {Animals ; Mice ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *T-Lymphocytes/immunology/metabolism ; Female ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics/pathology ; Interleukin-2/genetics/immunology/metabolism/administration & dosage ; Gene Knock-In Techniques ; Promoter Regions, Genetic/genetics ; Interleukin-12/genetics/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Male ; Transgenes/genetics ; Cell Line, Tumor ; },
abstract = {The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid tumours is limited by immunosuppression and antigen heterogeneity[1-3]. To overcome these barriers, 'armoured' CAR T cells, which secrete proinflammatory cytokines, have been developed[4]. However, their clinical application has been limited because of toxicity related to peripheral expression of the armouring transgene[5]. Here, we have developed a CRISPR knock-in strategy that leverages the regulatory mechanisms of endogenous genes to drive transgene expression in a tumour-localized manner. By screening endogenous genes with tumour-restricted expression, we have identified the NR4A2 and RGS16 promoters as promising candidates to support the delivery of cytokines such as IL-12 and IL-2 directly to the tumour site, leading to enhanced antitumour efficacy and long-term survival of mice in both syngeneic and xenogeneic models. This effect was concomitant with improved CAR T cell polyfunctionality, activation of endogenous antitumour immunity and a favourable safety profile, and was applicable in CAR T cells from patients.},
}

Animals
Mice
Humans
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*T-Lymphocytes/immunology/metabolism
Female
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics/pathology
Interleukin-2/genetics/immunology/metabolism/administration & dosage
Gene Knock-In Techniques
Promoter Regions, Genetic/genetics
Interleukin-12/genetics/immunology/metabolism
CRISPR-Cas Systems/genetics
Male
Transgenes/genetics
Cell Line, Tumor

@article {pmid40602043,
year = {2025},
author = {Gong, Y and Li, G and Meng, Y and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Development of a CRISPR/Cas13a-based electrochemiluminescence biosensing strategy for sensitive detection of α-synuclein oligomers in neurodegenerative diseases.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109038},
doi = {10.1016/j.bioelechem.2025.109038},
pmid = {40602043},
issn = {1878-562X},
mesh = {*alpha-Synuclein/blood/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Neurodegenerative Diseases/blood/diagnosis ; *Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; },
abstract = {In this study, we report a highly sensitive CRISPR/Cas13a-based electrochemiluminescence (ECL) biosensor for detecting α-synuclein oligomers, early biomarkers for neurodegenerative diseases. The system integrates aptamer recognition, T7 transcription, CRISPR/Cas13a cleavage, and EXPAR amplification. α-Synuclein binding triggers the release of the T7 promoter, leading to RNA production that activates Cas13a, initiating collateral cleavage and EXPAR, generating double-stranded DNA that interacts with [Ru(phen)2dppz][2+] to produce a measurable ECL signal. The sensor achieved an ultralow detection limit of 1.025 aM with high specificity and stability. In serum samples, recovery ranged from 95.2 % to 99.8 %, demonstrating strong accuracy. No interference was observed from unrelated proteins. The biosensor showed excellent reproducibility (intra-day RSD = 0.78 %, inter-day RSD = 2.86 %) and stable performance over 14 days. Compared to other existing methods, this strategy offers superior sensitivity and comparable dynamic range, making it highly suitable for clinical use. Although the assay requires multiple steps and approximately two hours, the tradeoff is justified by its performance. This work highlights the potential of combining CRISPR/Cas13a with ECL for ultra-sensitive biomarker detection in complex samples, supporting early diagnosis and monitoring of Parkinson's and Alzheimer's disease.},
}

*alpha-Synuclein/blood/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Neurodegenerative Diseases/blood/diagnosis
*Luminescent Measurements/methods
*Electrochemical Techniques/methods
Limit of Detection

@article {pmid40581010,
year = {2025},
author = {Zhang, S and Bu, G and Jin, H and Zhang, B},
title = {Nucleic acid-guided genome editing of plant carbohydrate pathways for functional crops supporting glycogen replenishment in athletes.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 4},
pages = {145574},
doi = {10.1016/j.ijbiomac.2025.145574},
pmid = {40581010},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *Glycogen/metabolism/biosynthesis ; *Ipomoea batatas/genetics/metabolism ; *Crops, Agricultural/genetics/metabolism ; *Carbohydrate Metabolism/genetics ; CRISPR-Cas Systems ; Starch/metabolism ; *Genome, Plant ; Humans ; },
abstract = {Nucleic acid-guided genome editing is transforming plant breeding by enabling precise modification of metabolic pathways to meet evolving food and nutritional needs. In this study, we applied multiplex CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) editing to improve Ipomoea batatas (sweet potato), a climate-resilient staple, for enhanced starch yield and customized digestibility suited for athletic performance nutrition. We targeted four key starch-related genes: AGPase (ADP-glucose pyrophosphorylase), GBSSI (granule-bound starch synthase I), SBEII (starch branching enzyme II), and GPT1 (glucose-6-phosphate translocator). Editing efficiency reached 85 to 92 % across multiple genome copies (homeologs), with no detectable off-target effects. The edited lines showed up to a 20 % increase in total starch and a fivefold reduction in amylose, which is the slowly digestible fraction of starch. This resulted in a high-amylopectin profile associated with rapid glucose release. Transcriptome analysis confirmed the metabolic shifts, including increased expression of GPT1 and sucrose synthase and reduced expression of invertase, which supports improved carbon allocation toward starch biosynthesis. In vitro digestibility and glycemic index (GI) assessments confirmed the functional impact of these traits for glycogen replenishment. Yield performance and drought resilience were retained, confirming agronomic viability. This study highlights the potential of CRISPR-based editing to develop climate-smart, nutritionally functional crops for both health-focused applications and global food security.},
}

*Gene Editing/methods
*Glycogen/metabolism/biosynthesis
*Ipomoea batatas/genetics/metabolism
*Crops, Agricultural/genetics/metabolism
*Carbohydrate Metabolism/genetics
CRISPR-Cas Systems
Starch/metabolism
*Genome, Plant
Humans

@article {pmid40544550,
year = {2025},
author = {Jia, L and Kang, K and Yan, W and Jin, Y and Xu, K and Wang, J and Wang, Y and Jiang, M and Yuan, W and Liu, S and Niu, L},
title = {17β-Estradiol point-of-care test by commercial pregnancy test strips based on target-triggered CRISPR/Cas12a cleavage activity.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109035},
doi = {10.1016/j.bioelechem.2025.109035},
pmid = {40544550},
issn = {1878-562X},
mesh = {*Estradiol/analysis/urine ; *CRISPR-Cas Systems ; Humans ; Female ; Pregnancy ; *Point-of-Care Testing ; *Pregnancy Tests/methods/instrumentation ; *Endodeoxyribonucleases/metabolism ; Chorionic Gonadotropin/chemistry ; Limit of Detection ; *Reagent Strips/analysis ; Biosensing Techniques/methods ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {17β-Estradiol (E2) serves as both a reproductive accelerator and growth promoter, yet its uncontrolled application induces severe endocrine dysfunction. Point-of-care testing (POCT) has emerged as a promising analytical platform for on-site E2 quantification, due to its inherent advantages in terms of sensitive, specific, cost-effective, and instrument-free. We developed a novel strategy combining the high collateral cleavage activity of CRISPR/Cas12a with the strong amplification power of dual-cycle reaction, translating target presence into a colorimetric signal on pregnancy test strip (PTS). In the presence of E2, the dual-cycle amplification was initiated to produce HP2-HP3 (Hairpin DNA 2-Hairpin DNA 3) duplexes. These duplexes then bound to the Cas12a-crRNA complex, activating its trans-cleavage activity. The activated Cas12a subsequently cleaved the ssDNA linkers in the MBs (magnetic beads)-ssDNA (single-stranded DNA)-hCG (human chorionic gonadotropin) conjugates, releasing hCG for quantitative E2 detection through strip color intensity measurement. By integrating CRISPR/Cas12a's high cleavage efficiency with dual-cycle amplification, a visible biosensor was developed, with a linear range of 1.0 × 10[-1]-200.0 pM and a detection limit of 0.0403 pM for E2. In conclusion, an E2 detection platform characterized by prominent sensitivity and convenience was established for monitoring spiked E2 in milk and urine, providing a significant reference for the POCT detection of other targets.},
}

*Estradiol/analysis/urine
*CRISPR-Cas Systems
Humans
Female
Pregnancy
*Point-of-Care Testing
*Pregnancy Tests/methods/instrumentation
*Endodeoxyribonucleases/metabolism
Chorionic Gonadotropin/chemistry
Limit of Detection
*Reagent Strips/analysis
Biosensing Techniques/methods
DNA, Single-Stranded/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40541695,
year = {2025},
author = {Ding, X and Lei, X and Yu, S},
title = {Split technology in sensors based on CRISPR/Cas12a system.},
journal = {Biotechnology advances},
volume = {83},
number = {},
pages = {108629},
doi = {10.1016/j.biotechadv.2025.108629},
pmid = {40541695},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Gene Editing ; DNA/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a system has become a popular tool for nucleic acid analysis in recent years due to its high specificity, sensitivity and programmability. Recently, split technology has been applied to the CRISPR/Cas12a system for activators, crRNA, reporter and Cas12a. As a result, dsDNA without PAM, short ssDNA less than 15 nucleotides, and RNA can be directly detected, which are beyond the target scope of the canonical CRISPR/Cas12a system. Label-free reporter with lower cost can be incorporated into sensors based on the CRISPR/Cas12a system. Logic circuits with multiple inputs and outputs can be constructed in cells. Therefore, split technology can expand the target scope, enhance crRNA stability, increase strategy programmability, and reduce detection cost for the CRISPR/Cas12a system. In this review, we focus on the advancements of split technology in sensors based on the CRISPR/Cas12a system. We also summarize the advantages brought by split technology and discuss the challenges and perspectives of sensors based on the CRISPR/Cas12a system.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Gene Editing
DNA/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40466204,
year = {2025},
author = {Zhou, Z and Li, Y and Wen, D and Zhang, X and Li, N and Shuai, J and Zhang, X and Zheng, X and Ban, Z and Farouk, A and Yu, X and Huang, J},
title = {A CRISPR-Cas13a powered electrochemical sensor based on reduced graphene oxide, polypyrrole and gold nanoparticles nanocomposites for PEDV detection.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {166},
number = {},
pages = {109019},
doi = {10.1016/j.bioelechem.2025.109019},
pmid = {40466204},
issn = {1878-562X},
mesh = {*Graphite/chemistry ; *Gold/chemistry ; *Pyrroles/chemistry ; *Porcine epidemic diarrhea virus/isolation & purification/genetics ; *Polymers/chemistry ; *Metal Nanoparticles/chemistry ; *Nanocomposites/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Animals ; *Electrochemical Techniques/methods ; Limit of Detection ; Swine ; Oxidation-Reduction ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen responsible for porcine epidemic diarrhea, which causes severe diarrhea and high mortality rates in neonatal piglets, leading to substantial economic losses in the swine industry. Therefore, there is an urgent need for rapid, sensitive, and accurate detection methods for PEDV. In this study, we develop a rapid, ultrasensitive electrochemical CRISPR-based biosensor for detecting PEDV with high sensitivity and specificity. The sensor integrates reduced graphene oxide-polypyrrole‑gold nanoparticle (rGO-PPy-AuNP) nanocomposites to enhance sensitivity and CRISPR-Cas13a for target-specific recognition. This dual-signal amplification strategy achieves an excellent analytical detection limit of 1.01 fg/mL and high linearity (R[2] = 0.9979) across a broad dynamic range (0.005-100,000 pg/mL) within 45 min, eliminating the need for nucleic acid amplification. By synergizing Cas13a-assisted signal enhancement with nanoparticle-mediated electron transfer, the biosensor outperforms conventional methods in sensitivity while maintaining excellent stability, and specificity, demonstrating strong potential for PEDV detection in clinical diagnostics.},
}

*Graphite/chemistry
*Gold/chemistry
*Pyrroles/chemistry
*Porcine epidemic diarrhea virus/isolation & purification/genetics
*Polymers/chemistry
*Metal Nanoparticles/chemistry
*Nanocomposites/chemistry
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Animals
*Electrochemical Techniques/methods
Limit of Detection
Swine
Oxidation-Reduction

@article {pmid40450408,
year = {2025},
author = {Einhaus, A and Krieger, A and Köhne, L and Rautengarten, B and Jacobebbinghaus, N and Saudhof, M and Baier, T and Kruse, O},
title = {Genome editing of epigenetic transgene silencing in Chlamydomonas reinhardtii.},
journal = {Trends in biotechnology},
volume = {43},
number = {8},
pages = {1961-1981},
doi = {10.1016/j.tibtech.2025.04.019},
pmid = {40450408},
issn = {1879-3096},
mesh = {*Chlamydomonas reinhardtii/genetics ; *Gene Editing/methods ; *Transgenes/genetics ; *Gene Silencing ; *Epigenesis, Genetic/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Eukaryotic microalgae are steadily advancing as promising green cell factories for sustainable modern biotechnology. However, one of the greatest hurdles to such use are the efficient transgene silencing mechanisms that drastically limit transgene expression levels and stability in engineered microalgal strains. Here, we used CRISPR/Cas9 to target multiple genes involved in epigenetic regulation in the model green microalga Chlamydomonas reinhardtii to identify key factors in epigenetic transgene silencing. Disruption of 11 candidate genes and subsequent systematic combination in double and triple knockout (KO) mutants, enabled a distinct reduction in transgene silencing and improved the stability of transgene expression compared with previously established strains. In addition, a split selectable marker system utilizing the Nostoc punctiforme DnaE split intein for dual-targeted genome editing was established. In summary, this work distinctly advances the biotechnological potential of C. reinhardtii and establishes a valuable mutant collection for further investigation of epigenetic regulation in green microalgae and potentially other eukaryotes.},
}

*Chlamydomonas reinhardtii/genetics
*Gene Editing/methods
*Transgenes/genetics
*Gene Silencing
*Epigenesis, Genetic/genetics
CRISPR-Cas Systems/genetics

@article {pmid40391806,
year = {2025},
author = {Chen, Z and Xue, J and Wang, Z and Sun, J and Cui, Y and Zhu, T and Yang, H and Li, M and Wu, B},
title = {Small RNA Toxin-Assisted Evolution of GC-Preferred ErCas12a for Enhanced Genome Targeting Range.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {29},
pages = {e17105},
doi = {10.1002/advs.202417105},
pmid = {40391806},
issn = {2198-3844},
support = {XDB0810000//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 32225002//National Natural Science Foundation of China/ ; 32170033//National Natural Science Foundation of China/ ; 32422001//National Natural Science Foundation of China/ ; ZDBS-LY-SM014//Key Research Program of Frontier Sciences/ ; KFJ-BRP-009//Biological Resources Program/ ; KFJ-BRP-017-58//Biological Resources Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics ; },
abstract = {CRISPR/Cas12a, a promising gene editing technology, faces limitations due to its requirement for a thymine (T)-rich protospacer adjacent motif (PAM). Despite the development of Cas12a variants with expanded PAM profiles, many genomic loci, especially those with guanine-cytosine (GC)-rich PAMs, have remained inaccessible. This study develops a small RNA toxin-aided strategy to evolve ErCas12a for targeting GC-rich PAMs, resulting in the creation of enhanced ErCas12a (enErCas12a). EnErCas12a demonstrates the ability to recognize GC-rich PAMs and target five times more PAM sequences than the wild-type ErCas12a. Furthermore, enErCas12a achieves efficient gene editing in both bacterial and mammalian cells at various sites with non-canonical PAMs, including GC-rich PAMs such as GCCC, CGCC, and GGCC, which are inaccessible to previous Cas12a variants. Moreover, enErCas12a effectively targets PAM sequences with a GC content exceeding 75% in mammalian cells, providing a valuable alternative to the existing Cas12a toolkit. Importantly, enErCas12a maintains high specificity at targets with canonical PAMs, while also demonstrating enhanced specificity at targets with non-canonical PAMs. Collectively, this work establishes enErCas12a as a promising tool for gene editing in both eukaryotes and prokaryotes.},
}

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

@article {pmid40253584,
year = {2025},
author = {Jo, DH and Jang, H and Cho, CS and Lee, SJ and Heo, JH and Kim, JA and Kim, SJ and Ryu, W and Park, CW and Kang, BC and Gee, HY and Sung, YH and Kim, HH and Kim, JH},
title = {Intravitreal adenine base editing of RS1 improves vision in a preclinical mouse model of retinoschisis.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {8},
pages = {3955-3967},
doi = {10.1016/j.ymthe.2025.04.021},
pmid = {40253584},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; *Retinoschisis/genetics/therapy ; Male ; *Adenine/metabolism ; Dependovirus/genetics ; *Eye Proteins/genetics ; Genetic Therapy/methods ; Genetic Vectors/genetics/administration & dosage ; Retina/metabolism/pathology ; Intravitreal Injections ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Vision, Ocular ; },
abstract = {Base editing offers high potential for treating genetic diseases, particularly those with limited treatment options. Retinoschisis, an X-linked retinal disease causing progressive vision loss, currently lacks effective therapies. We identified the c.422G>A (p.Arg141His) variant of the RS1 gene in six male patients with retinoschisis and generated a humanized mouse model harboring this variant, which mimicked the disease phenotype. By testing adenine base editors and single-guide RNAs, we identified an optimal combination of high editing efficiency and low bystander editing. Intravitreal injection of adeno-associated viral vectors encoding this adenine base editor achieved ∼40% editing efficiency in all retinal cells, restored retinal layer integrity, and preserved visual functions in 2-week-old male hemizygous mice. These mice exhibited retinal layer splitting at baseline, further validating the model. This study demonstrates a strategy for identifying effective base editing tools for clinical use through the preclinical evaluation of humanized mouse lines with patient-derived mutations and highlights their applicability in treating genetic diseases.},
}

Animals
Mice
*Gene Editing/methods
Disease Models, Animal
Humans
*Retinoschisis/genetics/therapy
Male
*Adenine/metabolism
Dependovirus/genetics
*Eye Proteins/genetics
Genetic Therapy/methods
Genetic Vectors/genetics/administration & dosage
Retina/metabolism/pathology
Intravitreal Injections
RNA, Guide, CRISPR-Cas Systems/genetics
*Vision, Ocular

@article {pmid40173114,
year = {2025},
author = {de Souza-Neto, RR and Cavalcante, LN and Carvalho, IGB and Curtolo, M and Benedetti, CE and Takita, MA and Wang, N and de Souza, AA},
title = {CRISPR/Cas9-Mediated Disruption of CsLIEXP1 Reveals Expansin as a Key Susceptibility Factor for Citrus Canker Disease.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {MPMI12240151R},
doi = {10.1094/MPMI-12-24-0151-R},
pmid = {40173114},
issn = {0894-0282},
abstract = {The Citrus sinensis LATERAL ORGAN BOUNDERIES 1 (CsLOB1) gene, which is directly induced by the Xanthomonas citri subsp. citri effector PthA4, functions as a transcription factor and citrus canker susceptibility (S) gene. Genome editing of CsLOB1 has been shown to confer resistance to citrus canker disease. Previous studies revealed that the citrus CsLOB1-INDUCED EXPANSIN 1 gene (CsLIEXP1) is highly and directly upregulated by CsLOB1 in Xanthomonas citri subsp. citri-infected plants. Because expansins are associated with cell wall loosening, potentially facilitating bacterial colonization, the CsLOB1-dependent activation of CsLIEXP1 is thought to contribute to canker symptoms and disease progression. Thus, CsLIEXP1 likely represents a critical canker susceptibility gene. In this study, we employed CRISPR/Cas9 to disrupt the function of CsLIEXP1 by modifying its corresponding coding region in Citrus sinensis cultivar 'Hamlin' and evaluated the postinfection responses of edited plants. DNA sequencing confirmed the edition of the CsLIEXP1-edited plant, which exhibited 26.47% of CsLIEXP1 edited sequences. Furthermore, CsLIEXP1 protein accumulation was reduced in CsLIEXP1-edited plants compared with the wild type when infected with X. citri. Leaves of edited plants inoculated with X. citri showed significantly fewer canker symptoms, with lesions limited to the site of bacterial inoculation and less pronounced cellular hypertrophy compared with control plants. Our results show that CsLIEXP1 is a citrus canker S gene that acts downstream of CsLOB1, thus providing new insights into plant-pathogen interactions. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.},
}

@article {pmid40119004,
year = {2025},
author = {Li, L and Bowling, S and Lin, H and Chen, D and Wang, SW and Camargo, FD},
title = {DARLIN mouse for in vivo lineage tracing at high efficiency and clonal diversity.},
journal = {Nature protocols},
volume = {20},
number = {8},
pages = {2319-2344},
pmid = {40119004},
issn = {1750-2799},
support = {32470700//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; },
mesh = {Animals ; Mice ; *Cell Lineage/genetics ; Single-Cell Analysis/methods ; *DNA Barcoding, Taxonomic/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Lineage tracing is a powerful tool to study cell history and cell dynamics during tissue development and homeostasis. An increasingly popular approach for lineage tracing is to generate high-frequent mutations at given genomic loci, which can serve as genetic barcodes to label different cell lineages. However, current lineage tracing mouse models suffer from low barcode diversity and limited single-cell lineage coverage. We recently developed the DARLIN mouse model by incorporating three barcoding arrays within defined genomic loci and combining Cas9 and terminal deoxynucleotidyl transferase (TdT) to improve editing diversity in each barcode array. We estimated that DARLIN generates 10[18] distinct lineage barcodes in theory, and enables the recovery of lineage barcodes in over 70% of cells in single-cell assays. In addition, DARLIN can be induced with doxycycline to generate stable lineage barcodes across different tissues at a defined stage. Here we provide a step-by-step protocol on applying the DARLIN system for in vivo lineage tracing, including barcode induction, estimation of induction efficiency, barcode analysis with bulk and single-cell sequencing, and computational analysis. The execution time of this protocol is ~1 week for experimental data collection and ~1 d for running the computational analysis pipeline. To execute this protocol, one should be familiar with sequencing library generation and Linux operation. DARLIN opens the door to study the lineage relationships and the underlying molecular regulations across various tissues at physiological context.},
}

Animals
Mice
*Cell Lineage/genetics
Single-Cell Analysis/methods
*DNA Barcoding, Taxonomic/methods
CRISPR-Cas Systems
Gene Editing/methods