@article {pmid41494892,
year = {2026},
author = {Wang, MR and Mu, W and Zhen, A and Kitchen, SG},
title = {CRISPR/Cas strategies to enhance CAR T-cell function and persistence via metabolic reprogramming.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.001},
pmid = {41494892},
issn = {1879-3096},
abstract = {While chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in various blood cancers, its full curative potential for other diseases has yet to be maximized. One key limiting factor is progressive T-cell exhaustion and differentiation over time, leading to the loss of the CAR-expressing cells. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) gene manipulation to enhance CAR T-cell therapy has revolutionized the field in recent years. In this review, we will examine the application of CRISPR/Cas aimed at improving CAR T-cell function and persistence to combat the issues of exhaustion and dysfunction, with a focus on metabolic reprogramming. Understanding current preclinical CRISPR/Cas strategies for modulating CAR T-cell metabolism is critical in advancing CAR-T therapies to clinical applications.},
}

@article {pmid41494604,
year = {2026},
author = {Zeng, Y and Zhao, G and Wu, S and Hu, B and Forn-Cuní, G and Knol, R and El Ghalbzouri, A and Snaar-Jagalska, E and Kros, A},
title = {CD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114598},
doi = {10.1016/j.jconrel.2025.114598},
pmid = {41494604},
issn = {1873-4995},
abstract = {Skin cancer is the third most common malignancy, with melanoma being the most challenging due to its resistance to current therapies. Gene editing technologies like CRISPR/Cas9 offer a promising strategy for targeting cancer-specific genes, but the efficient delivery of these tools to tumor sites remains a significant challenge. Lipid nanoparticles (LNPs) have emerged as the leading platform for gene editing tools due to their ability to protect and transport large payloads. To enhance the precision of gene editing in melanoma, we developed CD44-specific peptide-modified LNPs for targeted delivery of CRISPR/Cas9 mRNA and guide RNA against polo-like kinase 1 (sgPLK1). Our approach led to enhanced targeting and gene editing efficacy by specifically delivering CRISPR/Cas9 and sgPLK1 to melanoma tumor cells, resulting in significant inhibition of tumor growth in both in vitro and in vivo skin melanoma models. Moreover, this platform showed the capacity to reach metastatic melanoma in the brain and resulting in substantial suppression of tumor growth in brain metastasis models. We envision that this peptide-modification strategy could be further employed to improve the targeting capabilities and therapeutic outcomes of LNPs for CRISPR/Cas9-based gene editing, paving the way for more precise and effective cancer treatments.},
}

@article {pmid41492065,
year = {2026},
author = {Wang, Z and Wang, Y and Gao, H and Dai, J and Tang, N and Wang, Y and Ji, Q},
title = {Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.},
journal = {Nature microbiology},
volume = {11},
number = {1},
pages = {81-93},
pmid = {41492065},
issn = {2058-5276},
mesh = {*Thioredoxins/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Protein Binding ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA Cleavage ; DNA/metabolism ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.},
}

*Thioredoxins/metabolism/genetics
*CRISPR-Cas Systems
*Bacteriophages/genetics/enzymology/metabolism
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
Protein Binding
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
DNA Cleavage
DNA/metabolism
Escherichia coli/genetics/virology/metabolism

@article {pmid41489362,
year = {2026},
author = {Shin, J and Barrangou, R},
title = {Occurrence and applications of CRISPR-Cas systems in bifidobacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0170325},
doi = {10.1128/aem.01703-25},
pmid = {41489362},
issn = {1098-5336},
abstract = {Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.},
}

@article {pmid41488985,
year = {2026},
author = {Kim, K and Lee, J and Lee, N and Cho, BK},
title = {CRISPR-Based Approaches to Engineer Nonmodel Bacteria for Bioproduction and Biotherapeutics.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.5c00613},
pmid = {41488985},
issn = {1520-4995},
abstract = {Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.},
}

@article {pmid41488303,
year = {2025},
author = {Tsolakidou, PJ},
title = {CRISPR-Cas systems against carbapenem resistance: from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1725247},
pmid = {41488303},
issn = {1664-302X},
abstract = {Carbapenem-resistant Enterobacterales (CRE) pose a major global threat, driven by plasmid-borne carbapenemase genes such as bla KPC, bla NDM and bla OXA-48. CRISPR-Cas systems offer programmable strategies to selectively eliminate these resistance determinants. This mini-review summarizes recent advances in Cas9-based plasmid curing, RNA-targeting approaches such as Cas13a and Cas13d, and DNA-targeting Cas3-enhanced bacteriophage therapeutics that have entered early clinical evaluation. Particular attention is given to conjugative CRISPR-Cas9 plasmid systems, which enable targeted plasmid eradication without laboratory transformation and broaden the delivery toolbox beyond phage vectors. We further discuss major translational challenges, including delivery efficiency, phage host-range constraints, ecological risks of horizontal CRISPR dissemination, and off-target effects. Finally, we highlight emerging delivery platforms-outer membrane vesicles, lipid and polymeric nanoparticles, conjugative plasmids with containment circuits, and engineered live biotherapeutics-that may complement or overcome current limitations. Collectively, these developments illustrate the potential of CRISPR-based antimicrobials to augment traditional therapies through precise gene-level suppression of carbapenem resistance.},
}

@article {pmid41487293,
year = {2025},
author = {},
title = {Erratum: Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing [Corrigendum].},
journal = {Biologics : targets & therapy},
volume = {19},
number = {},
pages = {745-746},
doi = {10.2147/BTT.S585961},
pmid = {41487293},
issn = {1177-5475},
abstract = {[This corrects the article DOI: 10.2147/BTT.S326422.].},
}

@article {pmid41486484,
year = {2025},
author = {Boogari, M and Mohebbi, M and Hadidi, N},
title = {Genetically Engineered Probiotics: Design, Therapeutics, and Clinical Translation.},
journal = {Iranian biomedical journal},
volume = {29},
number = {6},
pages = {374-383},
doi = {10.61882/ibj.5197},
pmid = {41486484},
issn = {2008-823X},
abstract = {Genetically engineered probiotics aim to address transient colonization and the intra- and inter-subject variability that limit conventional probiotics. These strains utilizes CRISPR/Cas editing, programmable gene circuits, and biosensors in chassis such as E. coli Nissle 1917 and L. lactis. This narrative review summarizes the current engineering toolkits and standards (e.g., SEVA), chassis selection criteria, biocontainment strategies, and translational requirements under CMC/GMP frameworks and discusses regulatory considerations for clinical translation. Representative examples include IL-10-secreting L. lactis and phenylalanine-metabolizing strains for PKU (SYNB1618/SYNB1934), which illustrate pharmacodynamic target engagement and short-term preclinical safety. We outline clinical advancements in predefined pharmacodynamics, durability of function, monitoring shedding and HGT, and genomic-microbiome-informed patient stratification. Systems modeling approaches (GEM/ABM) are discussed as tools to guide rational design. GEPs offer programmable "sense-and-respond" therapeutics, with successful clinical adoption depending on durable efficacy, long-term safety, and clearly defined regulatory pathways.},
}

@article {pmid41484149,
year = {2026},
author = {Kremer, N and Mueller, F and Nguyen, H and Schulz, L and Popp, T and Artes, E and Wolters, J and Renner, M and Vetter, I and Maffini, S and Robles, MS and Musacchio, A and Bange, T},
title = {CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {132},
pmid = {41484149},
issn = {2041-1723},
support = {5041 140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Acetylation ; *src-Family Kinases/metabolism/genetics ; HEK293 Cells ; *DNA-Binding Proteins/metabolism/genetics ; Proteolysis ; Protein Processing, Post-Translational ; Ubiquitination ; Ubiquitin-Protein Ligases/metabolism ; CRISPR-Cas Systems ; },
abstract = {Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.},
}

*Cullin Proteins/metabolism/genetics
Humans
Acetylation
*src-Family Kinases/metabolism/genetics
HEK293 Cells
*DNA-Binding Proteins/metabolism/genetics
Proteolysis
Protein Processing, Post-Translational
Ubiquitination
Ubiquitin-Protein Ligases/metabolism
CRISPR-Cas Systems

@article {pmid41481841,
year = {2026},
author = {Jin, YM and Li, XD and Zhu, JK and Shao, CY and Huang, BB and Huang, HL and Wang, XW and Jiang, HB and Chen, W},
title = {Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70655},
doi = {10.1111/tpj.70655},
pmid = {41481841},
issn = {1365-313X},
support = {32470092//National Natural Science Foundation of China/ ; 32170108//National Natural Science Foundation of China/ ; 2024QL060//Ningbo Youth Leading Talent Project/ ; //Ningbo University Startup Funding/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Adenine/metabolism ; *Synechocystis/genetics ; *Cyanobacteria/genetics ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Anabaena/genetics ; },
abstract = {Cyanobacteria are photosynthetic prokaryotes with great potential in green biomanufacturing and basic research. Despite decades of pioneering achievements, the application of advanced genome editing tools, particularly CRISPR-based systems, has remained limited in cyanobacteria. In this study, we developed pCyABE, a new adenine base editor for efficient and precise A·T to G·C editing in cyanobacteria. This system utilizes a TadA-Cas9 nickase fusion and functions without double-strand breaks or donor templates. We demonstrated its high editing efficiency in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, highlighting its broad usability. pCyABE supports multiplex editing and enables start codon disruption for gene functional studies. Furthermore, this tool exhibits low off-target activity and can be effectively removed via sucrose counterselection. In conclusion, pCyABE provides a versatile and efficient genome editing platform that significantly expands the genetic toolbox for cyanobacterial research and biotechnology applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Adenine/metabolism
*Synechocystis/genetics
*Cyanobacteria/genetics
Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
*Anabaena/genetics

@article {pmid41481737,
year = {2026},
author = {Qi, S and Wei, L and Ding, Z and Zhong, F and Yang, S and Wu, L and Yang, X and Kang, B and Dan, M and Gan, J and Li, C and Su, X},
title = {Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eadu6505},
pmid = {41481737},
issn = {2375-2548},
mesh = {*Prealbumin/genetics/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; },
abstract = {Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the TTR gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based TTR editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for TTR gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.},
}

*Prealbumin/genetics/metabolism
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism/chemistry
Amyloid Neuropathies, Familial/genetics/therapy
HEK293 Cells

@article {pmid41481232,
year = {2026},
author = {Li, Y and Zhang, W and Wei, Z and Li, H and Liu, X and Zheng, T and Aziz, T and Xing, C and Meng, A and Wu, X},
title = {Stage- and tissue-specific gene editing using 4-OHT-inducible Cas9 in whole organism.},
journal = {The Journal of cell biology},
volume = {225},
number = {4},
pages = {},
pmid = {41481232},
issn = {1540-8140},
support = {#3258820001//National Natural Science Foundation of China/ ; #2023YFA1800300//National Key Research and Development Program of China/ ; 2018YFC1003304//National Key Research and Development Program of China/ ; #202302AO370011//Southwest United Graduate School/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Zebrafish/genetics/embryology ; *Tamoxifen/analogs & derivatives/pharmacology ; *CRISPR-Cas Systems/genetics ; Mice ; Female ; *CRISPR-Associated Protein 9/genetics/metabolism ; Germ Cells/metabolism ; Organ Specificity ; Humans ; Receptors, Estrogen/genetics/metabolism ; Animals, Genetically Modified ; },
abstract = {Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.},
}

Animals
*Gene Editing/methods
*Zebrafish/genetics/embryology
*Tamoxifen/analogs & derivatives/pharmacology
*CRISPR-Cas Systems/genetics
Mice
Female
*CRISPR-Associated Protein 9/genetics/metabolism
Germ Cells/metabolism
Organ Specificity
Humans
Receptors, Estrogen/genetics/metabolism
Animals, Genetically Modified

@article {pmid41479566,
year = {2025},
author = {Antipenko, ID and Venedyukhina, SA and Sorokina, NP and Kucherenko, IV and Smirnova, TS and Rogov, GN and Shkurnikov, MY},
title = {Whole-Genome Sequencing Uncovers Metabolic and Immune System Variations in Propionibacterium freudenreichii Isolates.},
journal = {Acta naturae},
volume = {17},
number = {4},
pages = {72-82},
pmid = {41479566},
issn = {2075-8251},
abstract = {Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.},
}

@article {pmid41479062,
year = {2026},
author = {Lee, NJ and Matsuoka, RL},
title = {CRISPR/Cas9-Based Mutagenesis Strategies for Efficient Biallelic Gene Inactivation and Consistent Phenotypic Detection in F0 Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2989},
number = {},
pages = {319-332},
pmid = {41479062},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Phenotype ; *Mutagenesis ; Alleles ; *Gene Silencing ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The zebrafish is a valuable animal model for investigating the genetic basis of vertebrate evolution, development, behavior, and regeneration. However, the existence of numerous gene paralogs in the zebrafish genome represents a major challenge, complicating functional genomic research using reverse-genetics approaches. To facilitate reverse genetics-based phenotypic screens, we recently presented simple methods that enable efficient induction of biallelic gene disruptions in F0 zebrafish, providing a rapid avenue for screening potential gene functions through consistent phenotypic detection. Here, we describe detailed protocols for these CRISPR/Cas9-based mutagenesis strategies to achieve highly effective biallelic gene inactivation in F0 zebrafish. The high consistency of these strategies, combined with a streamlined workflow, offers a robust phenotypic screening platform for a quick and reliable functional assessment of genes of interest, both individually and in a scalable manner. These strategies enhance the efficacy of successful F0 zebrafish phenotypic screening, thereby accelerating functional genetic studies using this powerful model organism.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems/genetics
Phenotype
*Mutagenesis
Alleles
*Gene Silencing
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41389602,
year = {2026},
author = {Kumari, P and Gupta, V and Chhikara, A and Dalal, J},
title = {Revolutionizing forensic DNA analysis: The potential of CRISPR-Cas9 technology in genetic investigations.},
journal = {Journal of forensic and legal medicine},
volume = {117},
number = {},
pages = {103047},
doi = {10.1016/j.jflm.2025.103047},
pmid = {41389602},
issn = {1878-7487},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Fingerprinting/methods ; *Forensic Genetics/methods ; *Gene Editing ; },
abstract = {The newest achievements in the field of molecular biology and gene-editing technologies have transformed the paradigm of forensic DNA analysis. However, there are still great difficulties in interpreting degraded, low-template, mixed genetic samples. The review critically evaluates the transformative potential of Clustered Regularly Interspaced Short Palindromic Repeats and an associated protein 9 (CRISPR-Cas9) as an accurate, effective, and cost-efficient system of genome-editing in the field of forensic science. Based on the evidence of the current literature, the paper critically analyzes the mechanisms of CRISPR-Cas9 activity, its RNA-guided specificity, dual-strand cleavage, and high-fidelity targeting, and compares its functionality with other standard methods like the STR and SNP profiling. The review also discusses more complex CRISPR-based diagnostic systems, such as SHERLOCK, DETECTR, and HOLMES that allow the analysis of DNA rapidly, without amplification, and in a portable format. Among major discoveries, there is the ability of CRISPR to increase the accuracy of DNA profiling, resolve mixture, recapitulate damaged genetic material, and reduce the possibility of contamination. In addition to genetic analysis, it has applications in forensic epigenetics, prediction of phenotypes, microbial forensics and environmental trace analysis. The review also covers the ethical, legal and governance implications of implementing CRISPR-based evidence in the judicial process especially in as far as data privacy; admissibility and equity of access are concerned. In general, CRISPR-Cas9 is a paradigm shift in forensic genomics, the one that has the potential to transform personal identification, reconstruction of the crime scene, and the interpretation of molecular evidence. Future efforts should focus on method validation, standardization, and ethical governance to ensure the responsible and sustainable implementation of this technology in forensic practice.},
}

Humans
*CRISPR-Cas Systems
*DNA Fingerprinting/methods
*Forensic Genetics/methods
*Gene Editing

@article {pmid41372414,
year = {2026},
author = {Habtewold, T and Lwetoijera, DW and Hoermann, A and Mashauri, R and Matwewe, F and Mwanga, R and Kweyamba, P and Maganga, G and Magani, BP and Mtama, R and Mahonje, MA and Tambwe, MM and Tarimo, F and Chennuri, PR and Cai, JA and Del Corsano, G and Capriotti, P and Sasse, P and Moore, J and Hudson, D and Manjurano, A and Tarimo, B and Vlachou, D and Moore, S and Windbichler, N and Christophides, GK},
title = {Gene-drive-capable mosquitoes suppress patient-derived malaria in Tanzania.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {442-448},
pmid = {41372414},
issn = {1476-4687},
mesh = {Tanzania/epidemiology ; Animals ; *Plasmodium falciparum/isolation & purification/genetics/drug effects/growth & development ; *Anopheles/genetics/parasitology ; Humans ; *Malaria, Falciparum/prevention & control/parasitology/transmission ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; Female ; Animals, Genetically Modified/genetics ; Male ; Child ; CRISPR-Cas Systems/genetics ; },
abstract = {Gene drive technology presents a transformative approach to combatting malaria by introducing genetic modifications into wild mosquito populations to reduce their vectorial capacity. Although effective modifications have been developed, these efforts have been confined to laboratories in the global north. We previously demonstrated that modifying Anopheles gambiae to express two exogenous antimicrobial peptides inhibits the sporogonic development of laboratory-cultured Plasmodium falciparum, with models predicting substantial contributions to malaria elimination in Africa when integrated with gene drive[1-3]. However, the effectiveness of this modification against genetically diverse, naturally circulating parasite isolates remained unknown. To address this critical gap, we adapted our technology for an African context by establishing infrastructural and research capacity in Tanzania, enabling the engineering of local A. gambiae under containment. Here we report the generation of a transgenic strain equipped with non-autonomous gene drive capabilities that robustly inhibits genetically diverse P. falciparum isolates obtained from naturally infected children. These genetic modifications were efficiently inherited by progeny when supplemented with Cas9 endonuclease provided by another locally engineered strain. Our work brings gene drive technology a critical step closer to application, providing a locally tailored and powerful tool for malaria eradication through the targeted dissemination of beneficial genetic traits in wild mosquito populations.},
}

Tanzania/epidemiology
Animals
*Plasmodium falciparum/isolation & purification/genetics/drug effects/growth & development
*Anopheles/genetics/parasitology
Humans
*Malaria, Falciparum/prevention & control/parasitology/transmission
*Gene Drive Technology/methods
*Mosquito Vectors/genetics/parasitology
Female
Animals, Genetically Modified/genetics
Male
Child
CRISPR-Cas Systems/genetics

@article {pmid41313207,
year = {2026},
author = {Wang, Y and Qin, Z and Wang, Q and Yang, Y and Gu, C and Yu, F and Wu, Y and Zhang, Lx},
title = {An RPA-CRISPR/Cas12a-based rapid and sensitive nucleic acid method for detection of Toxoplasma gondii in tissue and blood samples.},
journal = {Microbiology spectrum},
volume = {14},
number = {1},
pages = {e0155025},
pmid = {41313207},
issn = {2165-0497},
support = {231111111500//Key Research and Development Special Project of Henan Province of China/ ; 2022YFD1800200, 2023YFD1801200//National Basic Research Program of China/ ; },
mesh = {*Toxoplasma/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Protozoan/genetics ; *Toxoplasmosis/diagnosis/parasitology/blood ; Limit of Detection ; *Toxoplasmosis, Animal/diagnosis/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Toxoplasma gondii is a zoonotic pathogen that can infect humans and a wide range of warm-blooded animals, posing a significant threat to human health and the livestock industry. The development of a time-saving, highly sensitive, and specific method for the detection of T. gondii in tissue and blood samples is crucial to the monitoring, prevention, and control of toxoplasmosis. In this study, we evaluated the efficiency of a previously described method, termed REPORT, that integrates recombinase polymerase amplification with CRISPR/Cas12a for the detection of T. gondii nucleic acids. We evaluated the limit of detection (LOD) and specificity of the extended REPORT method using prepared target DNA in addition to tissue and blood samples. Furthermore, we validated the accuracy of T. gondii detection in clinical samples using the REPORT-based method in comparison with nested PCR based on the B1 gene. Sensitivity tests showed that the LOD of the REPORT-based fluorescence method and the lateral flow strip method were 3.7 copies /μL for target DNA, 3.1 tachyzoites/g for tissue samples, and five tachyzoites/mL for blood samples. Specificity tests suggested that the REPORT method had good specificity and did not cross-react with several common parasites. The method performed well for clinical DNA samples, demonstrating its ability for use in on-site detection.IMPORTANCEToxoplasma gondii can infect over 200 species of warm-blooded animals, including humans, posing not only a significant threat to public health systems but also causing substantial economic losses to the global livestock industry. Current diagnostic methods are slow, equipment-dependent, and impractical for field use. This study addresses these limitations by developing REPORT, a rapid, ultrasensitive nucleic acid test combining recombinase polymerase amplification and CRISPR/Cas12a. The REPORT detects T. gondii in tissue and blood samples within 1 h at low cost, requiring only a portable heater. Its visual results (fluorescence or test strips) enable on-site use without specialized training, achieving 100% accuracy versus nested PCR. With a sensitivity of 3.1 parasites per gram of tissue and five parasites per milliliter of blood, this method revolutionizes toxoplasmosis screening in resource-limited clinics, farms, and food safety inspections, empowering timely interventions to curb transmission and improve public health outcomes.},
}

*Toxoplasma/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Humans
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
DNA, Protozoan/genetics
*Toxoplasmosis/diagnosis/parasitology/blood
Limit of Detection
*Toxoplasmosis, Animal/diagnosis/parasitology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41225007,
year = {2026},
author = {Noh, H and Hashem, Z and Boms, E and Najafov, A},
title = {SIGLEC12 mediates plasma membrane rupture during necroptotic cell death.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {460-466},
pmid = {41225007},
issn = {1476-4687},
mesh = {*Necroptosis/genetics ; Humans ; Animals ; *Cell Membrane/metabolism/pathology ; Mice ; Protein Kinases/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Necroptosis is a form of lytic cell death that is overactivated during infections and in inflammatory pathologies[1]. NINJ1 was recently found to be a mediator of plasma membrane rupture (PMR) during pyroptosis, toxin-induced necrosis, apoptosis, and ferroptosis[2,3], but the mediator of PMR during necroptotic cell death remained unknown. Here, using a CRISPR-Cas9-based genome-wide knockout approach, we identify SIGLEC12 as a key mediator of necroptosis downstream of MLKL at the PMR step. Cells with knockdown or knockout of SIGLEC12 are defective in necroptosis-induced PMR and demonstrate ballooning morphology. During necroptosis, SIGLEC12 undergoes dephosphorylation, interacts with MLKL, forms cytosolic puncta and assembles into fibrils. Notably, SIGLEC12 is cleaved by TMPRSS4 during necroptosis to produce a 20-kDa fragment highly homologous to NINJ1, and this cleavage event is required and sufficient to induce PMR during necroptosis. A SIGLEC12 variant associated with cancer (Ser458Phe) and a variant found in the general human population (Arg528Trp) attenuate SIGLEC12 cleavage by TMPRSS4. Knockout of Siglec12 in mouse cells does not affect PMR, suggesting a species-specific role. Our identification of SIGLEC12 as a mediator of PMR expands our understanding of how programmed necrosis is executed and offers new approaches for targeting this proinflammatory form of cell death in human diseases.},
}

*Necroptosis/genetics
Humans
Animals
*Cell Membrane/metabolism/pathology
Mice
Protein Kinases/metabolism
Phosphorylation
CRISPR-Cas Systems
HEK293 Cells

@article {pmid40673390,
year = {2026},
author = {Guerra, F and De Rouck, S and Verhulst, EC},
title = {SYNCAS-mediated CRISPR-Cas9 genome editing in the Jewel wasp, Nasonia vitripennis.},
journal = {Insect molecular biology},
volume = {35},
number = {1},
pages = {48-55},
pmid = {40673390},
issn = {1365-2583},
support = {OCENW.M.22.140//Dutch Research Council (NWO)/ ; G035420N//Research Foundation Flanders (FWO)/ ; 101123162/ERC_/European Research Council/International ; },
mesh = {Animals ; *Wasps/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; Gene Knockout Techniques ; Insect Proteins/genetics ; },
abstract = {Genetic engineering is a formidable approach to studying biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to 10 times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques.},
}

Animals
*Wasps/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Female
Gene Knockout Techniques
Insect Proteins/genetics

@article {pmid39901548,
year = {2025},
author = {Yang, F and Wang, H and Fan, S and Qiu, H and Li, X and Shi, G and Li, Z and Luan, X and Wu, H},
title = {Advances in Synthetic Lethality in Potential Oncology Therapeutic Approaches.},
journal = {Current topics in medicinal chemistry},
volume = {25},
number = {15},
pages = {1868-1881},
pmid = {39901548},
issn = {1873-4294},
support = {ZR2022MB140//Natural Science Foundation of Shandong Province/ ; ZYQR201810168//National funding of postdoctoral innovative talents support program of Henan Province/ ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Agents/pharmacology/chemistry/therapeutic use ; *Synthetic Lethal Mutations/drug effects ; Molecular Targeted Therapy ; CRISPR-Cas Systems ; },
abstract = {Synthetic lethality represents a novel paradigm in molecular targeted cancer therapy. In synthetic lethality, perturbation of one gene alone does not hinder cell viability, yet simultaneous perturbation of both genes results in a loss of cellular viability. The presence of gene mutations in cancer cells, as opposed to normal cells, provides an opportunity for targeted therapies that mimic the effects of the second genetic mutation, enabling selective eradication of cancer cells. Recent advances in high-throughput screening technologies, such as CRISPR-Cas9 and RNA interference, have significantly enhanced the identification of synthetic lethal interactions, expanding the potential targets for therapeutic intervention. Challenges in exploiting synthetic lethality for cancer treatment include the complexities of tumor biology, limited comprehension of synthetic lethal interactions, drug resistance, and impediments in screening and clinical translation. Emerging strategies, such as combination therapies and novel drug designs, are being developed to overcome these obstacles. By virtue of its selective lethality towards cancer cells bearing specific genetic alterations, targeting synthetic lethal genes holds the promise to provide wider therapeutic windows compared to traditional cytotoxic chemotherapy. This review describes the current state of synthetic lethality applications in cancer treatment, encompassing both biological and methodological perspectives. It highlights the latest advancements in synthetic lethality with emerging interventional strategies. Furthermore, it explores future directions for research and clinical implementation, aiming to refine and expand the therapeutic potential of synthetic lethality in oncology.},
}

Humans
*Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Agents/pharmacology/chemistry/therapeutic use
*Synthetic Lethal Mutations/drug effects
Molecular Targeted Therapy
CRISPR-Cas Systems

@article {pmid41478990,
year = {2026},
author = {Hu, O and Provvido, A and Zhu, Y},
title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {361-370},
pmid = {41478990},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Gene Knockout Techniques/methods ; *Receptors, Interleukin-17/genetics ; Cell Line ; HEK293 Cells ; },
abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Gene Knockout Techniques/methods
*Receptors, Interleukin-17/genetics
Cell Line
HEK293 Cells

@article {pmid41478989,
year = {2026},
author = {Huo, Z and Tu, J and Shoemaker, R and Lee, DF and Zhao, R},
title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {343-359},
pmid = {41478989},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Cell Line ; *Tumor Suppressor Protein p53/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Human Embryonic Stem Cells/metabolism/cytology ; Exons ; Gene Knock-In Techniques/methods ; *Genetic Engineering/methods ; },
abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Cell Line
*Tumor Suppressor Protein p53/genetics
*Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Human Embryonic Stem Cells/metabolism/cytology
Exons
Gene Knock-In Techniques/methods
*Genetic Engineering/methods

@article {pmid41478695,
year = {2026},
author = {Zhang, H and Han, M and Pang, Z and Li, W and Li, X and Sun, B},
title = {The lldD lactate dehydrogenase is a determinant of lactic acid tolerance in Pichia kudriavzevii by pyruvate metabolism pathway.},
journal = {Food microbiology},
volume = {136},
number = {},
pages = {104989},
doi = {10.1016/j.fm.2025.104989},
pmid = {41478695},
issn = {1095-9998},
mesh = {*Pichia/genetics/metabolism/enzymology/drug effects ; *Lactic Acid/metabolism/pharmacology ; Fermentation ; *L-Lactate Dehydrogenase/genetics/metabolism ; Metabolic Networks and Pathways ; *Pyruvic Acid/metabolism ; *Fungal Proteins/genetics/metabolism ; Ethanol/metabolism ; CRISPR-Cas Systems ; Glucose/metabolism ; Gene Knockout Techniques ; },
abstract = {Lactic acid stress is common in traditional fermented foods. Pichia kudriavzevii owes its industrial prevalence to its superior acid tolerance, making deciphering its response mechanism imperative for sustainable fermentation processes. This work explored the remarkable acid tolerance of P. kudriavzevii, which can tolerate 80 g/L lactic acid. The key lactic acid tolerance gene lldD of P. kudriavzevii was determined through transcriptomic analysis. Importantly, this work knocked out the key gene lldD related to lactic acid tolerance for the first time by using the CRISPR-Cas9 technology. Meanwhile, the lldD knockout strain (pk-ΔlldD) was successfully constructed. There were significant differences between pk-ΔlldD and P. kudriavzevii in terms of tolerance to lactic acid, metabolism of lactic acid, utilization of glucose and ethanol production. In addition, the deletion of the gene lldD has a significant impact on the ABC transporter and metabolites of the amino acid metabolic pathway in P. kudriavzevii. In conclusion, this work provides a theoretical basis for engineering high acid-tolerant industrial yeast strains through targeted genetic modification. It helps enhance the stability of fermentation processes under lactic acid stress and ultimately lays a foundation for promoting efficient and low-loss production in the fermentation industry.},
}

*Pichia/genetics/metabolism/enzymology/drug effects
*Lactic Acid/metabolism/pharmacology
Fermentation
*L-Lactate Dehydrogenase/genetics/metabolism
Metabolic Networks and Pathways
*Pyruvic Acid/metabolism
*Fungal Proteins/genetics/metabolism
Ethanol/metabolism
CRISPR-Cas Systems
Glucose/metabolism
Gene Knockout Techniques

@article {pmid41477891,
year = {2026},
author = {Luo, G and Song, J and Fu, Y and Jiang, Y and Gao, Y and Zhong, Z and Li, L and Wei, Y and Jia, HR and Guo, L and Fu, T and Wu, Q and Tan, W},
title = {SPARK-seq: A high-throughput platform for aptamer discovery and kinetic profiling.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6780},
pages = {eadv6127},
doi = {10.1126/science.adv6127},
pmid = {41477891},
issn = {1095-9203},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; Kinetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; *Membrane Proteins/metabolism/genetics/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.},
}

*Aptamers, Nucleotide/chemistry/genetics
Kinetics
Humans
*High-Throughput Nucleotide Sequencing/methods
*Single-Cell Analysis/methods
*Membrane Proteins/metabolism/genetics/chemistry
RNA, Messenger/genetics
CRISPR-Cas Systems

@article {pmid41477825,
year = {2026},
author = {Song, LCT and Alker, ATP and Oromí-Bosch, A and Swartz, SE and Martinson, JNV and Arora, J and Wang, AM and Rovinsky, R and Smith, SJ and Pierce, EC and Deutschbauer, AM and Doudna, JA and Cress, BF and Rubin, BE},
title = {Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eaea1429},
pmid = {41477825},
issn = {2375-2548},
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; Escherichia coli/genetics ; Vibrio cholerae/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.},
}

*Gene Editing/methods
*Transposases/genetics/metabolism
Escherichia coli/genetics
Vibrio cholerae/genetics
*CRISPR-Cas Systems
*Genome, Bacterial
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41406662,
year = {2026},
author = {Schalper, KT and Yang, R and Guan, X and Zhang, J and Schreiber, D and Moon, J and Liu, C},
title = {Programmable CRISPR-mediated gold nanoparticle adhesion for visual colorimetric detection.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118319},
pmid = {41406662},
issn = {1873-4235},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {*Colorimetry/methods ; *Gold/chemistry ; *Biosensing Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis/virology ; *DNA, Viral/isolation & purification/genetics/analysis ; *Papillomaviridae/isolation & purification/genetics ; Female ; Limit of Detection ; DNA, Single-Stranded/chemistry ; },
abstract = {While the aggregation behavior of gold nanoparticles (AuNPs) has been extensively studied in biosensing, catalysis, and nanomedicine, their potential for programmable surface adhesion via tunable surface chemistry remains largely untapped. Here, a programmable CRISPR-mediated hydrophobic adhesion phenomenon using streptavidin-coated AuNPs functionalized with Cy5-ssDNA-biotin probes is introduced. Hydrophobic Cy5 moieties on the AuNP surface induce localized aggregation and strong adhesion to hydrophobic surfaces. This unique behavior was leveraged by coupling CRISPR-Cas12a-mediated ssDNA cleavage with Cy5-labeled ssDNA-coated AuNPs to develop a simple, visual-readout colorimetric assay for nucleic acid detection. When combined with recombinase polymerase amplification, the method achieved ultrasensitive detection of human papillomavirus (HPV) DNA down to 10 aM, without the need for complex instrumentation. The platform's clinical utility was validated by detecting HPV DNA in cervical swab samples, highlighting its promise for low-cost, sensitive, and accessible point-of-care diagnostics in resource-limited settings. Unlike conventional aggregation-dispersion systems, this platform introduces a fundamentally distinct signal transduction mechanism based on surface adhesion, defining a new modality within CRISPR-based colorimetric diagnostics and offering a simple, low-cost solution for point-of-care testing.},
}

*Colorimetry/methods
*Gold/chemistry
*Biosensing Techniques/methods
Humans
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
*Papillomavirus Infections/diagnosis/virology
*DNA, Viral/isolation & purification/genetics/analysis
*Papillomaviridae/isolation & purification/genetics
Female
Limit of Detection
DNA, Single-Stranded/chemistry

@article {pmid41402283,
year = {2025},
author = {Sivanandan, S and Leitmann, B and Lubeck, E and Sultan, MM and Stanitsas, P and Ranu, N and Ewer, A and Mancuso, JE and Phillips, ZF and Kim, A and Bisognano, JW and Cesarek, J and Ruggiu, F and Feldman, D and Koller, D and Sharon, E and Kaykas, A and Salick, MR and Chu, C},
title = {A pooled Cell Painting CRISPR screening platform enables de novo inference of gene function by self-supervised deep learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {77},
pmid = {41402283},
issn = {2041-1723},
mesh = {*Deep Learning ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pooled CRISPR screening enables large-scale interrogation of gene functions but typically measures simple phenotypes such as fitness. High-content methods like Perturb-seq extend dimensionality to transcriptomics but are costly and limited in scope. Optical pooled screening (OPS) combines pooled CRISPR screening with imaging to yield scalable, information-rich readouts, yet existing implementations remain pathway-specific. Here we describe an OPS-compatible Cell Painting platform that enables hypothesis-free reverse genetic screening through multiplexed morphological profiling. We validate this technique using a well-defined morphological gene set, compare classical image analysis to self-supervised learning methods using a mechanism-of-action library, and perform discovery screening with a druggable genome library. By combining rich morphological data with deep learning, gene networks emerge without the need for target-specific biomarkers, leading to unbiased discovery of gene functions.},
}

*Deep Learning
Humans
*CRISPR-Cas Systems/genetics
Gene Regulatory Networks
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41381248,
year = {2026},
author = {Adamopoulos, PG and Athanasopoulou, K and Scorilas, A},
title = {A versatile type VI CRISPR-based approach for targeted m[6]A demethylation in mRNAs.},
journal = {Genome research},
volume = {36},
number = {1},
pages = {169-182},
pmid = {41381248},
issn = {1549-5469},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Adenosine/analogs & derivatives/metabolism ; HeLa Cells ; AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Demethylation ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {Epitranscriptomics, a rapidly evolving field mainly driven by massive parallel sequencing technologies, explores post-transcriptional RNA modifications. N [6]-methyladenosine (m[6]A) has emerged as the most prominent and dynamically regulated modification in human mRNAs, being implicated in the regulation of diverse biological processes, including spermatogenesis, heat shock response, ultraviolet-induced DNA damage response and maternal mRNA clearance. Despite the recognized significance of m[6]A in mRNA regulation, limited studies have focused on the targeted and efficient manipulation of this modification in mRNAs. Here, we present Dem6A-Vec, an "all-in-one" plasmid vector designed for site-specific m[6]A demethylation in human mRNAs. Dem6A-Vec integrates the expression of a catalytically inactive RfxCas13d fused to the m[6]A demethylase ALKBH5 and a U6-driven customizable guide RNA in a single construct, simplifying experimental workflows and enhancing targeting efficiency. Using nanopore direct RNA sequencing, we identify high-confident m[6]A sites in HeLa cells, which serve as targets for Dem6A-Vec. We validate the targeted demethylation of m[6]A sites in the EEF2 and RRAGA genes using the established SELECT-qPCR method, confirming the impacts on mRNA stability and highlighting the tool's precision and versatility. The presented approach is implemented in multiple mRNA sites with diverse methylation stoichiometries, underscoring its adaptability to various transcriptomic contexts. This study provides a robust and scalable method for investigating the functional roles of m[6]A modifications, offering a transformative platform for advancing epitranscriptomic research and potential therapeutic applications.},
}

Humans
*RNA, Messenger/genetics/metabolism
*Adenosine/analogs & derivatives/metabolism
HeLa Cells
AlkB Homolog 5, RNA Demethylase/genetics/metabolism
Demethylation
*CRISPR-Cas Systems
*RNA Processing, Post-Transcriptional

@article {pmid41380236,
year = {2026},
author = {Yin, Z and Yin, H and Zhou, Y and Liu, H},
title = {CRISPR/Cas13a-mediated photoelectrochemical-colorimetric dual-mode biosensor for RNA N-acetyltransferase 10 detection based on Bi2O2S/Ag2S and laccase-like nanozyme.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118298},
doi = {10.1016/j.bios.2025.118298},
pmid = {41380236},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Laccase/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; *Acetyltransferases/isolation & purification/analysis/chemistry ; Humans ; CRISPR-Cas Systems ; Limit of Detection ; Bismuth/chemistry ; },
abstract = {N-Acetyltransferase 10 (NAT10) is a crucial protein that catalyzes RNA acetylation modification and plays a significant role in biological activities. Accurate detection of NAT10 is of great importance in clinical testing and drug development. To achieve this goal, a novel biosensing platform was developed for NAT10 detection, relying on an RNA acetylation-inhibited CRISPR/Cas13a system, a Bi2O2S/Ag2S type-II heterojunction, and a laccase-mimetic nanocatalyst, using a photoelectric-colorimetric dual-mode strategy. Based on the catalysis effect of laccase-mimetic nanocatalyst, its substrates (hydroquinone and 2,4-dichlorophenol) were oxidized to form p-benzoquinone and 2,4-dichloroquinone, respectively. This oxidation process not only impaired the electron-donating ability of hydroquinone but also induced the coupling of 2,4-dichloroquinone with 4-antipyrine to generate a red product-enabling both photoelectrochemical and colorimetric detection of the NAT10 protein. The biosensor exhibited wide linear ranges of 0.1-1000 μg/L (photoelectrochemical mode) and 1-1000 μg/L (colorimetric mode), and the low detection limit of 0.056 and 0.348 μg/L for the two modes, respectively. Additionally, this biosensor was used to investigate the inhibitory effects of plasticizers and phosphorus-containing flame retardants on NAT10. Furthermore, molecular docking simulations were employed to explore the underlying inhibition mechanism. The developed biosensor not only provided a novel detection technique for NAT10, but also offers an alternative method for NAT10 inhibitor screening and a new tool for evaluating the ecotoxicological effects of organic pollutants.},
}

*Biosensing Techniques/methods
Laccase/chemistry
Electrochemical Techniques/methods
Colorimetry/methods
*Acetyltransferases/isolation & purification/analysis/chemistry
Humans
CRISPR-Cas Systems
Limit of Detection
Bismuth/chemistry

@article {pmid41330302,
year = {2026},
author = {Xu, W and Lin, Y and Huang, Z and Li, Y and Lu, Y and Liu, M and Cui, S and Zhang, T and Shi, N and Sheng, Y and Hu, J},
title = {Split proximity circuit initiated CRISPR-Cas12a system profiling exosomal surface proteins for early cancer detection.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118280},
doi = {10.1016/j.bios.2025.118280},
pmid = {41330302},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/diagnosis/blood/genetics ; *Early Detection of Cancer/methods ; Female ; Epithelial Cell Adhesion Molecule/genetics/isolation & purification ; *Biomarkers, Tumor/genetics/blood ; Limit of Detection ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Early diagnosis of breast cancer is critical for improving prognosis, but traditional methods have limitations. Herein, we propose an SPC-CRISPR system for the sensitive and specific detection of multiple breast cancer exosomal proteins without prior exosome isolation. This system couples CRISPR system with an enzyme-free amplification method to achieve dual-signal amplification. SPC-CRISPR is based on a split proximity circuit (SPC) that triggers catalytic hairpin assembly (CHA), converting protein signals on the surface of exosomes into nucleic acid signals, and the CRISPR-Cas12a system enabling further signal amplification and output. The system targets phosphatidylserine (PS), MUC1, and EpCAM on exosomes: Tim4-modified magnetic beads capture PS-expressing exosomes, and dual-aptamers recognize MUC1 and EpCAM, enabling SPC assembly and subsequent amplification. In buffer and cell-derived exosomes, the SPC-CRISPR system showed a detection limit of 10 particles/μL (R[2] = 0.990). Clinical tests utilizing merely 1 μL of serum samples successfully distinguished breast cancer patients from healthy donors (AUC = 0.9778, accuracy = 91.23 %), detected stage 0 breast cancer patients against healthy controls (accuracy = 92.59 %), and differentiated metastatic from non-metastatic cases (p < 0.001). The combination of high sensitivity, minimal sample requirements, and an exosome isolation-free workflow positions the SPC-CRISPR system as a promising tool for the clinical early detection and classification of breast cancer, with broader applicability to other cancers by swapping the corresponding aptamers.},
}

Humans
*Exosomes/chemistry/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Breast Neoplasms/diagnosis/blood/genetics
*Early Detection of Cancer/methods
Female
Epithelial Cell Adhesion Molecule/genetics/isolation & purification
*Biomarkers, Tumor/genetics/blood
Limit of Detection
Mucin-1/genetics
Aptamers, Nucleotide/chemistry

@article {pmid41318543,
year = {2025},
author = {Pernaci, C and Johnson, A and Gillette, S and Warden, AS and McCormick, C and Weiser-Novak, S and Ramirez, G and Broersma, EH and Mishra, P and Sivakumar, A and Cherqui, S and Coufal, NG},
title = {Microgliopathy as a primary mediator of neuronal death in models of Friedreich's Ataxia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {81},
pmid = {41318543},
issn = {2041-1723},
support = {TRAN1-13983//California Institute for Regenerative Medicine (CIRM)/ ; EDUC2-08388//California Institute for Regenerative Medicine (CIRM)/ ; R01NS135162//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {*Friedreich Ataxia/pathology/genetics/metabolism ; Animals ; *Microglia/pathology/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Frataxin ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Cell Death ; Mitochondria/metabolism/pathology ; *Neurons/pathology/metabolism ; Purkinje Cells/pathology/metabolism ; Trinucleotide Repeat Expansion ; Iron/metabolism ; Male ; Female ; White Matter/pathology ; CRISPR-Cas Systems ; },
abstract = {Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.},
}

*Friedreich Ataxia/pathology/genetics/metabolism
Animals
*Microglia/pathology/metabolism
Humans
Induced Pluripotent Stem Cells/metabolism
Mice
Frataxin
Disease Models, Animal
Iron-Binding Proteins/genetics/metabolism
Cell Death
Mitochondria/metabolism/pathology
*Neurons/pathology/metabolism
Purkinje Cells/pathology/metabolism
Trinucleotide Repeat Expansion
Iron/metabolism
Male
Female
White Matter/pathology
CRISPR-Cas Systems

@article {pmid41277417,
year = {2026},
author = {Choi, MY and Li, C and Choi, JH and Choi, JW},
title = {Intracellular biosensors by functional nanomaterial-integrated CRISPR technologies for real-time molecular sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {1},
pages = {58-70},
doi = {10.1039/d5cc05016b},
pmid = {41277417},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; Gene Editing ; Nanotechnology ; },
abstract = {CRISPR technology, originally developed as a genome-editing tool, has recently emerged as a powerful platform for intracellular biosensing. By harnessing the programmability and target specificity of CRISPR-associated proteins, such as Cas9, Cas12, and Cas13, researchers have engineered biosensors capable of detecting a wide array of intracellular signals, including nucleic acids, non-coding RNAs, and small-molecule metabolites. This review discusses the recent advancements in CRISPR-powered biosensors for real-time, dynamic monitoring of cellular processes and molecular events. Particular focus is given to the integration of nanotechnology, which plays a crucial role in enhancing the delivery efficiency, signal amplification, and sensor stability. Nanomaterials such as gold nanoparticles, quantum dots, DNA nanostructures, and upconversion nanoparticles have been strategically employed to improve the intracellular transport of CRISPR components, facilitate signal readouts, and enable multimodal sensing in complex cellular environments. Additionally, we delve into how CRISPR-nanotechnology hybrids can be adapted for multiplex analysis and single-cell resolution. This review also addresses the current challenges in intracellular biosensing, including precise delivery, biocompatibility, and long-term monitoring, and outlines future directions for the application of these systems in precision medicine, synthetic biology, and advanced therapeutic monitoring. Through the convergence of gene-editing systems and nanotechnology, CRISPR-based intracellular biosensors are anticipated to revolutionize next-generation diagnostic and therapeutic strategies.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Nanostructures/chemistry
Gene Editing
Nanotechnology

@article {pmid41261774,
year = {2026},
author = {Sun, W and Zhu, Y and Zhang, X and Meng, X and Xia, K and Zhang, X and Han, X and Pan, W and Guo, J and Li, J and Zhang, H},
title = {A mortise-tenon joint system facilitates precise targeted DNA insertion and replacement in rice.},
journal = {Molecular plant},
volume = {19},
number = {1},
pages = {36-47},
doi = {10.1016/j.molp.2025.11.006},
pmid = {41261774},
issn = {1752-9867},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Mutagenesis, Insertional/methods ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; DNA, Plant/genetics ; },
abstract = {Precise and scarless DNA insertion and replacement represent two of the major challenges in plant genome editing. Numerous tools have been developed, including homology-directed repair-dependent tools and prime-editing-mediated systems. However, none has fully resolved these challenges. In this study, we develop the "mortise-tenon joint system" (MT), a novel strategy that enables precise and efficient targeted DNA insertion and replacement. By leveraging the APOBEC-Cas9-uracil DNA glycosylase/(apurinic or apyrimidinic site) lyase within our previously reported APOBEC-Cas9 fusion-induced deletion system, which performs single-strand cleavage on the non-target strand and double-strand cleavage on the target strand, we generated a unique "mortise" structure, consisting of double-strand breaks with single or double non-complementary 5' overhangs. We further designed "tenons," double-stranded DNA donors containing 5' sticky ends precisely matching the 5' overhangs of the mortises. The end-capture interaction between mortises and tenons facilitates precise targeted insertion and replacement, achieving frequencies of 16.30%-59.47% across seven tested targets using 21-85 bp donor inserts in rice. If long DNA donors with sticky ends complementary to the mortise structure can be generated, the MT system may enable highly precise targeted insertion and replacement of large DNA fragments.},
}

*Oryza/genetics
*Gene Editing/methods
*Mutagenesis, Insertional/methods
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
DNA, Plant/genetics

@article {pmid41148632,
year = {2026},
author = {Jin, Z and Yang, Y and Chen, C and Zhang, Z and Ren, Q and Cui, Z and Huang, CZ and Zuo, H},
title = {Gene silencing regulated by aggregates of Corn aptamer at 3' UTR of mRNA.},
journal = {Nanoscale horizons},
volume = {11},
number = {1},
pages = {232-242},
doi = {10.1039/d5nh00510h},
pmid = {41148632},
issn = {2055-6764},
mesh = {*RNA, Messenger/genetics/chemistry/metabolism ; *3' Untranslated Regions/genetics ; *Gene Silencing ; *Aptamers, Nucleotide/chemistry/genetics ; Humans ; CRISPR-Cas Systems ; *Zea mays/genetics/chemistry ; },
abstract = {Gene therapy, as a cutting-edge approach for disease intervention, relies heavily on advancements in gene silencing techniques. For instance, CRISPR-Cas9 has emerged as a leading gene-editing tool due to its ability to introduce precise cuts at specific genomic loci, enabling targeted gene insertion, deletion, or modification. In this study, we developed a simple and effective gene silencing strategy by introducing a nucleic acid self-assembly module into the 3' untranslated region (UTR) of mRNA. This module demonstrated significant gene silencing efficacy in eukaryotic cells through the formation of RNA aggregates. To systematically investigate its regulatory mechanism on translation efficiency through the formation of higher-order RNA structures, we quantitatively analyzed both mRNA and protein expression levels. Furthermore, our modular 3' UTR sequences can be integrated with classical 5' UTR elements (e.g., TOP sequences) to construct a multidimensional post-transcriptional regulatory network. This technology expands the diversity of existing UTR element libraries and offers a reservoir of programmable regulatory elements for applications in synthetic biology. It enables the construction of orthogonal combinations of multidimensional elements, tailored to specific gene expression regulation needs.},
}

*RNA, Messenger/genetics/chemistry/metabolism
*3' Untranslated Regions/genetics
*Gene Silencing
*Aptamers, Nucleotide/chemistry/genetics
Humans
CRISPR-Cas Systems
*Zea mays/genetics/chemistry

@article {pmid41121528,
year = {2026},
author = {Horsley, N and Sythoff, AVB and Delgado, M and Liu, S and Cabernard, C},
title = {CrisprBuildr: an open-source application for CRISPR-mediated genome engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {1},
pages = {},
pmid = {41121528},
issn = {2160-1836},
support = {R35GM148160/NH/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; *Genome, Insect ; *Genetic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.},
}

Animals
*Drosophila melanogaster/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Software
*Genome, Insect
*Genetic Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41477711,
year = {2025},
author = {Rozov, SM and Deineko, EV},
title = {[Recombinase-Based Engineering of Plant Genomes in the Era of Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {6},
pages = {873-890},
doi = {10.7868/S3034555325060016},
pmid = {41477711},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *DNA Nucleotidyltransferases/genetics/metabolism ; *Recombinases/genetics/metabolism ; *Plants/genetics ; DNA Transposable Elements ; },
abstract = {The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.},
}

*Gene Editing/methods
*Genome, Plant
*CRISPR-Cas Systems
*DNA Nucleotidyltransferases/genetics/metabolism
*Recombinases/genetics/metabolism
*Plants/genetics
DNA Transposable Elements

@article {pmid41475352,
year = {2025},
author = {Martinho, C and Hoshino, M and Raphalen, M and Bukhanets, V and Kerur, A and Bogaert, KA and Luthringer, R and Coelho, SM},
title = {Efficient CRISPR-Cas genome editing in brown algae.},
journal = {Cell reports methods},
volume = {},
number = {},
pages = {101273},
doi = {10.1016/j.crmeth.2025.101273},
pmid = {41475352},
issn = {2667-2375},
abstract = {Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATEUPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.},
}

@article {pmid41475348,
year = {2025},
author = {Gao, X and Zhu, K and Zhang, W and Wang, L and Wang, L and Hua, L and Niu, T and Qin, B and Yu, X and Zhu, H and Cui, S},
title = {RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2025.12.005},
pmid = {41475348},
issn = {1097-4164},
abstract = {RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.},
}

@article {pmid41475278,
year = {2025},
author = {Kolanchi, P and Saminathan, N and Selvaraj, D and Krishnamoorthy, A and Palanivelu, K and Aruchalam, A},
title = {Bioprocess and genetic advances enhancing Beauveria bassiana biocontrol efficacy.},
journal = {Microbial pathogenesis},
volume = {211},
number = {},
pages = {108272},
doi = {10.1016/j.micpath.2025.108272},
pmid = {41475278},
issn = {1096-1208},
abstract = {Beauveria bassiana is a widely exploited entomopathogenic fungus that has emerged as a central component of ecologically sustainable pest management. Recent years have witnessed rapid progress across its biological understanding, technological development, and application potential. This review synthesizes contemporary advances spanning infection biology, secondary metabolite biosynthesis, strain development, bioprocess engineering, formulation science, and genetic improvement. At the molecular level, multi-omics studies have elucidated the coordinated regulation of surface adhesion, cuticular penetration, host immune modulation, dimorphic transitions, and toxin production, revealing gene networks that govern virulence, stress tolerance, and ecological adaptation. These insights have informed improved strategies for strain isolation and high-throughput phenotypic screening, enabling the selection of isolates with enhanced pathogenicity, environmental robustness, and endophytic competence. Parallel advances in solid-state and submerged fermentation, supported by agro-industrial substrates and data-driven optimization, have strengthened large-scale production of infective propagules with consistent quality. Such gains are further reinforced by modern formulation approaches, including oil-based dispersions, encapsulation systems, nanoemulsions, and seed-coating technologies, which collectively improve spore stability, persistence, and delivery under heterogeneous field conditions. More recently, CRISPR/Cas-based genome editing and pathway engineering have opened new avenues for precision enhancement of virulence traits, metabolic output, and abiotic stress resilience. Despite these achievements, the broader adoption of B. bassiana remains constrained by variable field performance, slower speed of action relative to chemical insecticides, strain-dependent efficacy, and regulatory and quality-control challenges. By integrating fundamental biology with technological innovation and practical limitations, this review provides a coherent framework for advancing B. bassiana from laboratory optimization to reliable field implementation, underscoring its promise as a next-generation, environmentally aligned biocontrol agent in modern agriculture.},
}

@article {pmid41474879,
year = {2025},
author = {Cheng, M and Wang, Y and Lin, W and Ye, J and Wu, M and Xiang, B and Liu, L and Sun, B},
title = {A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c05722},
pmid = {41474879},
issn = {1520-6882},
abstract = {Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to Mycoplasma pneumoniae detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.},
}

@article {pmid41048051,
year = {2026},
author = {Huang, LT and Gao, RJ and Zhang, D and Nian, C and Martzke, W and Shapiro, AMJ and Kin, T and Tahamtani, Y and Lynn, FC},
title = {Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells.},
journal = {Disease models & mechanisms},
volume = {19},
number = {1},
pages = {},
doi = {10.1242/dmm.052376},
pmid = {41048051},
issn = {1754-8411},
support = {5-SRA-2020-1059-S-B, 3-COE-2022-1103-M-B//Juvenile Diabetes Research Foundation United States of America/ ; ASD-173663//Institute of Nutrition, Metabolism and Diabetes/ ; 5238 BIOM//Michael Smith Health Research BC/ ; IGAP//BC Children's Hospital/ ; 5-SRA-2020-1059-S-B//Breakthrough T1D Canada/ ; 3-COE-2022-1103-M-B//Breakthrough T1D Canada/ ; ASD-173663/CAPMC/CIHR/Canada ; //BC Children's Hospital/ ; 6563//National Science and Engineering Research Council of Canada/ ; },
mesh = {Humans ; *Insulin-Secreting Cells/cytology/metabolism ; *Antigens, CD19/metabolism ; Biomarkers/metabolism ; *Cell Separation/methods ; Human Embryonic Stem Cells/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Insulin/metabolism ; C-Peptide/metabolism ; Cell Differentiation ; },
abstract = {Stem cell-derived β-cells (SCβ-cell) are a renewable and scalable alternative to cadaveric islets as a cell-replacement therapy for type 1 diabetes (T1D). However, heterogeneity within SCβ-cell cultures remains problematic for graft safety and function. Magnetic selection of SCβ-cells expressing a unique cell-surface marker may help deplete undesirable cell types and facilitate functional maturation. Here, we explored the transmembrane glycoprotein CD19 as a potential cell-surface marker for the enrichment of insulin-expressing SCβ-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of insulin (INS) coding sequence exon 2 in human embryonic stem cells (hESCs). We developed and optimized a magnetic-activated cell sorting protocol for CD19-mScarlet-expressing cells, forming enriched SCβ-cell clusters with improved glucose-stimulated C-peptide secretion. This strategy holds promise to facilitate large-scale production of functional SCβ-cells for disease modeling and cell-replacement therapy.},
}

Humans
*Insulin-Secreting Cells/cytology/metabolism
*Antigens, CD19/metabolism
Biomarkers/metabolism
*Cell Separation/methods
Human Embryonic Stem Cells/cytology/metabolism
CRISPR-Cas Systems/genetics
Insulin/metabolism
C-Peptide/metabolism
Cell Differentiation

@article {pmid41316351,
year = {2025},
author = {Qin, Z and Wang, Y and Sun, M and Wang, Q and Duan, J and Gu, C and Zhang, X and Yu, F and Wu, Y and Xu, H and Li, J and Zhang, L},
title = {Development of a field-deployable RPA-CRISPR/Cas12a assay for the detection of Cyclospora cayetanensis in human feces.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {4},
pmid = {41316351},
issn = {1756-3305},
support = {232102110088//Henan Province Scientific and Technological Project/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Project of Henan Province/ ; },
mesh = {*Cyclospora/isolation & purification/genetics ; Humans ; *Feces/parasitology ; *Cyclosporiasis/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Cyclospora is an emerging intestinal pathogenic protozoan transmitted through foodborne and waterborne routes. At least 19 countries in the world have recorded outbreaks of cyclosporiasis, mainly associated with the consumption of contaminated fresh agricultural products. The lack of a sensitive immediate test is one of the major obstacles to the rapid diagnosis of cyclosporiasis. The target interference mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been adapted into versatile and efficient genome manipulation and disease-curing technologies, while also being promising for point-of-care testing (POCT) applications. It can serve as an excellent rapid and specific detection tool.

METHODS: The recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system were combined to develop a detection method for C. cayetanensis (termed RECCT-Cay) via visual observation of fluorescent readings under blue light and field diagnosis using lateral flow strip (LFS) biosensors.

RESULTS: The detection limit of the established RECCT-Cay was 7 copies/μL. Under simulated clinical conditions, the detection limit was 30 oocysts per gram of stool. At the same time, the established detection platform can distinguish C. cayetanensis from the closely related Eimeria spp. The results of our constructed assay were compared with nested PCR, and the detection results of 30 clinical stool samples were consistent, with three samples positive for C. cayetanensis. Based on the RECCT-Cay detection principle, a portable suitcase-sized device has been designed, which can conduct rapid on-site detection of clinical samples.

CONCLUSIONS: The RECCT-Cay platform features rapid speed, high sensitivity, and the capability for field detection, making it a promising tool for use in remote areas.},
}

*Cyclospora/isolation & purification/genetics
Humans
*Feces/parasitology
*Cyclosporiasis/diagnosis/parasitology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/genetics/metabolism
Biosensing Techniques/methods
*Molecular Diagnostic Techniques/methods

@article {pmid41474623,
year = {2025},
author = {Banh, DV and Goldberg, GW and Marraffini, LA},
title = {Phage induction of Staphylococcus aureus pathogenicity islands promotes the CRISPR-Cas adaptive immune response.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116776},
doi = {10.1016/j.celrep.2025.116776},
pmid = {41474623},
issn = {2211-1247},
abstract = {Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements carrying virulence genes that spread upon infection by helper phages that induce their transfer. Staphylococci also carry type II and III CRISPR-Cas systems that mount an adaptive immune response against phages through the acquisition of spacer sequences from viral genomes, directing Cas nucleases to their targets. Whether and how SaPIs and CRISPR interact with each other during helper phage infection is not known. Here we report that, as a result of the packaging of incomplete helper phage genomes into SaPI particles, defective viral DNA delivered into new hosts stimulates spacer acquisition in both CRISPR types. Once immunized, staphylococci target the helper phage and prevent SaPI mobilization. Our work reveals an unexpected synergy between CRISPR-Cas systems and SaPIs that enhances antiphage immunity and could favor the retention of beneficial elements within the population.},
}

@article {pmid41474367,
year = {2026},
author = {Rasool, HMH and Gong, X and Jin, Y and Liu, M and Yanyan, W and Qu, M and Anxiang, Z and Lingling, X and Li, Z and Zhou, J and Chen, Q},
title = {Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71397},
doi = {10.1096/fj.202503610R},
pmid = {41474367},
issn = {1530-6860},
support = {2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024BEG-02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2024312885//Comprehensive Control Technology For Brucellosis and Hydatic Disease in Xinjiang/ ; CAAS-ZDRW202410//The Agricultural Science and Technology Innovation Program (ASTIP)/ ; },
mesh = {*Brucella/genetics/isolation & purification ; Animals ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Cattle ; Fluorescence ; Limit of Detection ; Humans ; },
abstract = {Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella, including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL-3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time-consuming nature of assays, making them unsuitable for large-scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost-effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA-CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella. Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA-CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA-CRISPR/Cas12a-F and 10 copies/μL for RPA-CRISPR/Cas12a-LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA-CRISPR/Cas12a assay showed high concordance with classical quantitative real-time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.},
}

*Brucella/genetics/isolation & purification
Animals
*Brucellosis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
Sensitivity and Specificity
Cattle
Fluorescence
Limit of Detection
Humans

@article {pmid41472258,
year = {2025},
author = {Gladue, DP and O'Mahony, A},
title = {CRISPR Treatments for AI-Designed Synthetic Viruses: Rapid Programmable Countermeasures for Emerging and Engineered Viruses.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472258},
issn = {1999-4915},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Viral ; *Artificial Intelligence ; *Synthetic Biology/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Viruses/genetics ; Animals ; Genetic Engineering/methods ; },
abstract = {The convergence of artificial intelligence and synthetic biology is innovating and accelerating the design of novel viral genomes, expanding both therapeutic opportunities and dual-use risk. This review articulates a countermeasure strategy for emerging and engineered viruses leveraging the programmable CRISPR modality. Building on mounting in vitro and in vivo evidence that Cas9 degrades DNA viruses (e.g., Orthopoxviruses, HSV-1, ASFV), while Cas13 targets RNA viral genomes (e.g., Influenza A, Dengue, RSV), both leading to reduced viremia, diminished disease burden, and alleviated symptoms. Here, we outline a rapid-response pipeline to position CRISPR-based countermeasures in translational and pandemic-response frameworks, linking real-time sequencing to AI-assisted gRNA selection and multiplexed cassette design to achieve viral targeting efficacy. To minimize resistance and off-target risk, we emphasize multi-gRNA cocktails, continuous genomic surveillance, and adaptive gRNA rotation. We also propose governance mechanisms, such as pre-cleared gRNA repositories, transparent design logs, standardized off-target/safety screening, and alignment with evolving nucleic-acid-synthesis screening frameworks to enable emergency deployment while preserving security. Furthermore, compressing the time from sequence to treatment and complementary to vaccines and small-molecule antivirals, CRISPR represents a technologically agile and strategically essential capability to combat both natural outbreaks and AI-enabled biothreats. Collectively, programmable CRISPR antivirals represent an auditable, rapidly adaptable foundation for next-generation biodefense preparedness.},
}

Humans
*CRISPR-Cas Systems
Genome, Viral
*Artificial Intelligence
*Synthetic Biology/methods
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Viruses/genetics
Animals
Genetic Engineering/methods

@article {pmid41472250,
year = {2025},
author = {Tan, C and Xing, S and Xie, X and Chen, X and Liu, X and Wang, W and Liu, L and Zhou, X and Wu, J and Li, C},
title = {Development and Application of a Rapid Field Detection Technology for DENV-2 Based on the HUDSON Nucleic Acid Extraction-Free/RT-RAA/CRISPR-Cas12a System.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472250},
issn = {1999-4915},
support = {2024YFC2607800//Chunxiao-Li/ ; },
mesh = {*Dengue Virus/genetics/isolation & purification/classification ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; Animals ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue fever has become a major global public health challenge due to its rapidly in-creasing incidence. Rapid on-site detection of dengue virus (DENV) is critical for early diagnosis, timely patient isolation, and outbreak control. In this study, dengue virus serotype 2 (DENV-2), the predominant strain circulating in tropical and subtropical regions, was selected as the target pathogen. We established a one-tube rapid detection assay that integrates the HUDSON nucleic acid extraction-free protocol, reverse transcription recombinase-aided amplification (RT-RAA), and CRISPR/Cas12a-mediated trans cleavage activity. The method achieved a detection limit of 1 × 10[2] copies/μL for simulated infected samples and exhibited no cross-reactivity with other DENV serotypes (DENV-1, DENV-3, DENV-4) or with other arboviruses, including Zika, Japanese encephalitis, yellow fever, and chikungunya viruses. The assay demonstrated high sensitivity and specificity across various sample types, including mosquitoes, rodents, blood, and cultured cells, with results consistent with quantitative PCR (qPCR). Requiring only basic equipment such as a water bath, the system enables on-site detection of DENV-2 within 1 h. This simple, cost-effective, and reliable assay provides a practical tool for field-based DENV-2 surveillance and supports effective public health responses in resource-limited settings.},
}

*Dengue Virus/genetics/isolation & purification/classification
*Dengue/diagnosis/virology
*CRISPR-Cas Systems
Humans
Sensitivity and Specificity
Animals
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41472204,
year = {2025},
author = {Dong, J and He, X and Bao, S and Wei, Z},
title = {Diagnostic Methods for Bovine Coronavirus: A Review of Recent Advancements and Challenges.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472204},
issn = {1999-4915},
support = {KJZC-2024-15//Gansu Provincial Department of Agriculture and Rural Affairs Science and Technology Support Project/ ; },
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/genetics/isolation & purification ; *Cattle Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Coronavirus Infections/diagnosis/veterinary/virology ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.},
}

Animals
Cattle
*Coronavirus, Bovine/genetics/isolation & purification
*Cattle Diseases/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*Coronavirus Infections/diagnosis/veterinary/virology
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
CRISPR-Cas Systems

@article {pmid41472049,
year = {2025},
author = {Slukin, PV and Fursov, MV and Volkov, DV and Sizova, AA and Detushev, KV and Dyatlov, IA and Fursova, NK},
title = {Diversity of CRISPR-Cas Systems Identified in Urological Escherichia coli Strains.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472049},
issn = {2076-2607},
support = {075-15-2025-525 of 30.05.2025.//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Type I-E and I-F CRISPR-Cas systems were identified in 237 E. coli strains isolated from patients with urinary tract infections (UTIs) between 2004 and 2019. The strains were classified into nine distinct groups (I-IX) based on the presence or absence of cas genes and repeat regions (RRs). Within the type I-E systems, two sequence variants were identified, distinguished by polymorphisms in the casB, cas3, cas7, cas5, and cas6 genes. The direct repeats (DRs) also differed, with I-E-associated RRs ranging from 26 to 32 bp and I-F-associated RRs consistently being 28 bp. We identified 762 unique spacers (29-35 bp in length) across the strain collection, while the number of spacers per strain varied from 1 to 47, and potential DNA targets were determined for 65 spacers, targeting 38 bacteriophage genomes, 19 plasmids, and 8 cas genes of the I-F type CRISPR-Cas system. Multilocus sequence typing (MLST) revealed 68 sequence types and 24 clonal complexes (CCs), with the most prevalent being ST131, CC10, CC69, CC405, CC14, CC38, CC73, and CC648. Significant correlations were observed between specific phylogroups/CCs, the type of CRISPR-Cas system present, and distinct profiles of virulence and antibiotic resistance genes.},
}

@article {pmid41472024,
year = {2025},
author = {Yi, M and Hu, Y and Fan, B and Pan, Y and Pan, B and Wang, J and Liu, Z},
title = {Advances in Novel Detection Technologies for Occult Hepatitis B Virus Infection: Building an Ultra-Sensitive Barrier for Transfusion Safety.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472024},
issn = {2076-2607},
abstract = {Occult hepatitis B virus infection (OBI), characterized by extremely low viral loads and the persistent intrahepatic presence of cccDNA, poses a profound challenge to global public health security. With a prevalence ranging from 0.06% to over 15% in different donor populations, OBI maintains a risk of transmission and can progress to hepatocellular carcinoma. Its prevention and control have long been limited by the sensitivity constraints of conventional detection methods, highlighting the urgent need for more sensitive diagnostic innovations. Emerging technologies offer distinct breakthroughs: ddPCR facilitates absolute quantification; CRISPR-Cas systems coupled with isothermal amplification enable rapid, point-of-care testing; third-generation sequencing resolves viral integration and mutations; and nanomaterials enhance the signal detection. This review synthesises advancements in OBI diagnostic technologies and provides a comparative overview of their strengths, limitations, and transfusion safety implications, as well as their potential applications in blood transfusion. Recommendations are also proposed to inform the advancement of OBI risk control in blood transfusion and to guide the development of novel diagnostic technologies, particularly relevant to regions with high HBV endemicity, such as China.},
}

@article {pmid41251053,
year = {2026},
author = {Shrestha, M and Kim, Y and Park, S and Jiang, HL and Lee, JY and Kim, D and Jeong, JH},
title = {eVLP-Mediated Cas9 Delivery for Preventing IBMIR in Islet Transplantation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {1},
pages = {e08128},
doi = {10.1002/smll.202508128},
pmid = {41251053},
issn = {1613-6829},
support = {//National Research Foundation of Korea/ ; RS-2025-02303064//Korean government/ ; RS-2024-02507183//Ministry of Health and Welfare/ ; 22A0205L1//Ministry of Health and Welfare/ ; 23A0205L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/adverse effects/methods ; Thromboplastin/metabolism/genetics ; Plasminogen Activator Inhibitor 1/metabolism/genetics ; Rats ; Diabetes Mellitus, Experimental/therapy ; Male ; *Inflammation/prevention & control ; *CRISPR-Associated Protein 9/metabolism ; Mice ; Islets of Langerhans ; *CRISPR-Cas Systems/genetics ; },
abstract = {Islet transplantation is a promising strategy for effective β-cell replacement in patients with type 1 diabetes. However, its success is hindered significantly by instant blood-mediated inflammatory reaction (IBMIR), which leads to rapid graft loss. IBMIR is triggered when the transplanted islets come in contact with blood, activating the coagulation cascade, complement pathways, and innate immune responses. Tissue factor (TF), abundantly expressed on the islet surface, initiates the coagulation cascade, leading to thrombin formation, platelet activation, and neutrophil infiltration. Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in IBMIR by inhibiting fibrinolysis and causing ischemic injury in the graft. TF and PAI-1 contribute significantly to IBMIR, thus making them critical targets for genetic interventions to prevent IBMIR. In this study, an engineered virus-like particle (eVLP)-mediated Cas9 nuclease is employed to knock out TF and PAI-1 genes in rat islets. TF and PAI-1 expression are effectively downregulated without inducing any off-target effects or without compromising the viability and functionality of the islets. Streptozotocin-induced diabetic mice transplanted with TF- and PAI-1-knockout islets exhibited improved glycemic control and a significant reduction in the plasma levels of thrombin-antithrombin (TAT) complex and complement component 3a (C3a), indicating the successful inhibition of IBMIR post-transplantation.},
}

Animals
*Islets of Langerhans Transplantation/adverse effects/methods
Thromboplastin/metabolism/genetics
Plasminogen Activator Inhibitor 1/metabolism/genetics
Rats
Diabetes Mellitus, Experimental/therapy
Male
*Inflammation/prevention & control
*CRISPR-Associated Protein 9/metabolism
Mice
Islets of Langerhans
*CRISPR-Cas Systems/genetics

@article {pmid40899441,
year = {2026},
author = {Patel, UA and Shi, MY and Kazan, JM and Nixon, KCJ and Ran, X and Nair, SN and Huang, O and Song, L and Aparnathi, MK and He, MY and Bakhtiari, M and Krishnan, R and Hessenow, RK and Philip, V and Ketela, T and Jendrossek, V and Hakem, R and He, HH and Kridel, R and Lok, BH},
title = {CRISPR Screen Identifies HDAC3 as a Novel Radiosensitizing Target in Small Cell Lung Cancer.},
journal = {Molecular cancer therapeutics},
volume = {25},
number = {1},
pages = {183-195},
pmid = {40899441},
issn = {1538-8514},
support = {U01 CA253383/CA/NCI NIH HHS/United States ; UL1 TR000457/TR/NCATS NIH HHS/United States ; //Terry Fox Research Institute (TFRI)/ ; //Canada Foundation for Innovation (CFI)/ ; //Cancer Research Society (CRS)/ ; //Canadian Institutes of Health Research (CIHR)/ ; U01CA253383//National Cancer Institute (NCI)/ ; UL1TR00457//Clinical and Translational Science Center, Weill Cornell Medicine (CTSC)/ ; GRK 2762/1//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; Animals ; *Small Cell Lung Carcinoma/genetics/pathology/radiotherapy/drug therapy ; *Histone Deacetylases/genetics/metabolism ; Mice ; *Lung Neoplasms/genetics/pathology/radiotherapy ; *Radiation-Sensitizing Agents/pharmacology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Radiation Tolerance/genetics/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Acrylamides ; Phenylenediamines ; },
abstract = {Small cell lung cancer (SCLC) is an aggressive malignancy, with most patients presenting with prognostically poor extensive-stage disease. Limited progress in standard care stresses the urgent need for novel therapies. Radiotherapy offers some survival benefit for selected patients with SCLC but could be enhanced with radiosensitizers. In this study, we identify HDAC3 as a novel radiosensitizing target in SCLC using a CRISPR knockout screen and demonstrate its efficacy and mechanism. SBC5 cells were transduced with a custom EpiDrug single-guide RNA library and treated with ionizing radiation (IR) to identify radiosensitizing genes. HDAC3 emerged as a candidate and was validated through genetic knockdown and pharmacologic inhibition (RGFP966) in multiple SCLC cell lines. Both approaches enhanced radiosensitivity, as shown by cell viability (dose modification factor10 = 1.14-1.69) and clonogenic assays (dose modification factor10 = 1.16-1.41). We assessed changes in chromatin accessibility by assay for transposase-accessible chromatin using sequencing and IR-induced DNA damage and repair using γH2AX foci detection, double-strand break (DSB) repair assays, and immunoblotting of repair proteins. HDAC3-deficient cells exhibited increased chromatin accessibility, greater IR-induced DSBs, and impaired repair capacity, resulting in persistent DNA damage. This repair defect sensitized cells to PARP inhibitors, for which combining RGFP966 with olaparib or talazoparib produced additive to synergistic effects. In SCLC xenograft models, HDAC3 knockdown or RGFP966, combined with IR, achieved significant tumor growth inhibition. Collectively, we identified HDAC3 as a novel radiosensitizing target in SCLC. Its functional loss increased the generation and persistence of IR-induced DNA DSBs, effectively sensitizing SCLC cell lines and xenografts to IR, providing a potential radiosensitization strategy to treat SCLC.},
}

Humans
Animals
*Small Cell Lung Carcinoma/genetics/pathology/radiotherapy/drug therapy
*Histone Deacetylases/genetics/metabolism
Mice
*Lung Neoplasms/genetics/pathology/radiotherapy
*Radiation-Sensitizing Agents/pharmacology
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Radiation Tolerance/genetics/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats
Acrylamides
Phenylenediamines

@article {pmid41471222,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {From Methylomes to CRISPR Epigenetic Editing: New Paths in Antibiotic Resistance.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/pathogens14121267},
pmid = {41471222},
issn = {2076-0817},
mesh = {*Gene Editing/methods ; *Epigenesis, Genetic ; *Bacteria/genetics/drug effects ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; DNA Methylation ; *Epigenome ; *Drug Resistance, Microbial/genetics ; Gene Expression Regulation, Bacterial ; Epigenome Editing ; },
abstract = {Antibiotic resistance (AR) has long been interpreted through the lens of genetic mutations and horizontal gene transfer. Yet, mounting evidence suggests that epigenetic regulation, including DNA and RNA methylation, histone-like proteins, and small non-coding RNAs, plays a similarly critical role in bacterial adaptability. These reversible modifications reshape gene expression without altering the DNA sequence, enabling transient resistance, phenotypic heterogeneity, and biofilm persistence under antimicrobial stress. Advances in single-molecule sequencing and methylome mapping have uncovered diverse DNA methyltransferase systems that coordinate virulence, efflux, and stress responses. Such epigenetic circuits allow pathogens to survive antibiotic exposure, then revert to susceptibility once pressure subsides, complicating clinical treatment. Parallel advances in CRISPR-based technologies now enable direct manipulation of these regulatory layers. CRISPR interference (CRISPRi) and catalytically inactive dCas9-fused methyltransferases can silence or reactivate genes in a programmable, non-mutational manner, offering a new route to reverse resistance or sensitize pathogens. Integrating methylomic data with transcriptomic and proteomic profiles further reveals how epigenetic plasticity sustains antimicrobial tolerance across environments. This review traces the continuum from natural bacterial methylomes to engineered CRISPR-mediated epigenetic editing, outlining how this emerging interface could redefine antibiotic stewardship. Understanding and targeting these reversible, heritable mechanisms opens the door to precision antimicrobial strategies that restore the effectiveness of existing drugs while curbing the evolution of resistance.},
}

*Gene Editing/methods
*Epigenesis, Genetic
*Bacteria/genetics/drug effects
*CRISPR-Cas Systems
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Humans
DNA Methylation
*Epigenome
*Drug Resistance, Microbial/genetics
Gene Expression Regulation, Bacterial
Epigenome Editing

@article {pmid41471176,
year = {2025},
author = {Bhowmik, S and Rivu, S and Bari, ML and Ahmed, S},
title = {Genome Mining of Cronobacter sakazakii in Bangladesh Reveals the Occurrence of High-Risk ST83 and Rare ST789 Lineages.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/pathogens14121220},
pmid = {41471176},
issn = {2076-0817},
support = {BIO-34//University Grant Commission, Bangladesh/ ; },
mesh = {Bangladesh/epidemiology ; Humans ; *Cronobacter sakazakii/genetics/isolation & purification/classification/pathogenicity ; *Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/epidemiology ; Virulence Factors/genetics ; Infant ; Food Microbiology ; Plasmids/genetics ; Infant, Newborn ; Whole Genome Sequencing ; Phylogeny ; Infant Formula/microbiology ; },
abstract = {Cronobacter sakazakii is a foodborne pathogen of major concern due to its link with severe neonatal infections through powdered infant formula (PIF). However, its genomic epidemiology in Bangladesh remains uncharacterized. We report the first whole-genome analysis of three isolates from PIF. Two isolates (S41_PIFM and S44_RUTF) belonged to ST83, a lineage repeatedly associated with neonatal meningitis, septicemia, and persistence in PIF production environments, while the third (S43_TF) represented ST789, a recently described and rare lineage of unknown pathogenic potential. Pan-genome and comparative analyses identified 39 virulence determinants, 19 antimicrobial-resistance genes, and diverse mobile genetic elements. ST83 isolates harbored plasmid replicons IncFII(pCTU2) and pESA2, while the ST789 isolate carried insertion sequence ISKpn34, indicating horizontal gene transfer potential. All strains encoded I-E CRISPR-Cas systems. The detection of globally recognized high-risk ST83 clones alongside the novel ST789 lineage highlights emerging public health risks. This study provides the first genomic insights into C. sakazakii in Bangladesh and underscores the urgent need for genomic surveillance and strengthened food safety monitoring to protect infant health in low- and middle-income countries.},
}

Bangladesh/epidemiology
Humans
*Cronobacter sakazakii/genetics/isolation & purification/classification/pathogenicity
*Genome, Bacterial
*Enterobacteriaceae Infections/microbiology/epidemiology
Virulence Factors/genetics
Infant
Food Microbiology
Plasmids/genetics
Infant, Newborn
Whole Genome Sequencing
Phylogeny
Infant Formula/microbiology

@article {pmid41468306,
year = {2026},
author = {Wang, Y and Li, Y and Li, J and Li, M and Qiu, X},
title = {Silencing of optogenetic and chemogenetic transgenes in human iPSCs involves promoter methylation and methylation-independent mechanisms.},
journal = {Epigenetics},
volume = {21},
number = {1},
pages = {2606983},
doi = {10.1080/15592294.2025.2606983},
pmid = {41468306},
issn = {1559-2308},
mesh = {Humans ; *Promoter Regions, Genetic ; *Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; *DNA Methylation ; Optogenetics ; *Transgenes ; *Gene Silencing ; CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Editing ; Chemogenetics ; },
abstract = {The transplantation of neural progenitor cells derived from induced pluripotent stem cells (iPSCs) has therapeutic potential for the treatment of neurological diseases. However, the functional integration of transplanted iPSC-derived neurons into host neural networks remains controversial. Optogenetic and chemogenetic tools offer the means to assess such integration. However, constructing modifiable iPSC-derived neurons requires efficient gene editing. Here, we used CRISPR/Cas9 (targeting the AAVS1 safe harbor) and PiggyBac transposon systems to insert optogenetic and chemogenetic receptors (ChR2/hM4Di) into human iPSCs. While both systems successfully integrated genes into the genomes of HEK293T cells and iPSCs, receptor expression was detected only in HEK293T cells. Bisulfite sequencing revealed extensive methylation of the TRE3G BI promoter (95.3-98.2%) in iPSCs, in contrast to low methylation (5.9%) in HEK293T cells. For PiggyBac, the methylation of CMV/EF1α promoters in iPSCs exhibited integration site-dependent variability (0-95.2%). Notably, even hypomethylated clones failed to show gene expression, suggesting that additional regulatory mechanisms, such as histone modifications or chromatin remodeling, may contribute to transcriptional silencing. Differentiation into neural stem cells does not reverse methylation nor restore protein expression. Our findings demonstrate that the CRISPR/Cas9 and PiggyBac systems enable the integration of optochemical receptor genes into iPSCs. However, promoter methylation or other epigenetic and non-epigenetic gene-silencing mechanisms could pose barriers to efficient protein expression from the integrated transgene in iPSCs.},
}

Humans
*Promoter Regions, Genetic
*Induced Pluripotent Stem Cells/metabolism/cytology
HEK293 Cells
*DNA Methylation
Optogenetics
*Transgenes
*Gene Silencing
CRISPR-Cas Systems
DNA Transposable Elements
Gene Editing
Chemogenetics

@article {pmid41467478,
year = {2026},
author = {Niazian, M and de Ronne, M and Beauchamp, CJ and Belzile, F and Torkamaneh, D},
title = {CRISPR-induced knockouts reveal a dual role for the soybean NFR5α gene in symbiotic nitrogen fixation and root hair development.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70143},
pmid = {41467478},
issn = {1940-3372},
support = {6548//Genome Canada/ ; 337003//Fonds de recherche du Québec - Nature et technologies (FRQNT)/ ; },
mesh = {*Glycine max/genetics/growth & development/microbiology ; *Nitrogen Fixation/genetics ; *Plant Roots/growth & development/genetics ; *Symbiosis/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Root Nodulation/genetics ; },
abstract = {Nitrogen fixation in soybeans, facilitated by symbiotic interactions with rhizobia, is a cornerstone of sustainable agriculture, reducing reliance on synthetic fertilizers. However, the efficiency of symbiotic nitrogen fixation (SNF) varies due to natural genetic variation in SNF-related genes. Our study underscores the pivotal role of the GmNFR5α gene not only in nodulation but also in root hair development, which is crucial for effective nutrient uptake and plant yield. Through detailed genetic analyses and clustered regularly interspaced short palindromic repeats (CRISPR)-based manipulations, we identified and characterized multiple knockout mutants, notably GmNFR5α-KO and combined GmNFR5α+GmROP6-KO, which exhibited significant reductions in root hair density and nodulation. These phenotypic changes correspond with the downregulation of key root hair development genes such as TTG, RHD1, RHD2, and KJK, establishing a clear link between GmNFR5α function and root hair formation. The potential of leveraging these genetic insights to improve nitrogen fixation in legumes and introduce SNF capabilities into cereal crops could revolutionize crop fertilization strategies, offering a sustainable solution to global agricultural challenges.},
}

*Glycine max/genetics/growth & development/microbiology
*Nitrogen Fixation/genetics
*Plant Roots/growth & development/genetics
*Symbiosis/genetics
CRISPR-Cas Systems
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Plant Root Nodulation/genetics

@article {pmid41270510,
year = {2026},
author = {Li, J and Li, X and Li, X and Xu, W and Yuan, L and Shi, H and Lei, Z and Li, N and Wei, Y and Hua, J},
title = {CRISPR/Cas9-generated CD46-knockout spermatogonial stem cells reveal mechanisms of BVDV-induced reproductive dysfunction in male livestock.},
journal = {Veterinary microbiology},
volume = {312},
number = {},
pages = {110807},
doi = {10.1016/j.vetmic.2025.110807},
pmid = {41270510},
issn = {1873-2542},
mesh = {Animals ; Male ; Goats ; Cattle ; CRISPR-Cas Systems ; *Diarrhea Viruses, Bovine Viral/physiology/pathogenicity ; *Bovine Virus Diarrhea-Mucosal Disease/virology ; *Adult Germline Stem Cells/virology ; Reproduction ; Gene Knockout Techniques ; *Spermatogonia/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV) is a major viral pathogen that affects ruminants, resulting in significant economic losses due to issues such as immunosuppression, reproductive disorders, and growth retardation. Bulls infected with this virus may become infertile within a few months and can transmit the virus to susceptible cattle during mating. However, the mechanism of BVDV impairing the reproductive function of male livestock is not clear, as there is no suitable cell model. This study used spermatogonial stem cells(SSCs) from cattle and goats as research materials to explore the mechanism by which BVDV affects the reproductive function of male livestock. The results of this study indicate that both cytopathic (cp) and noncytopathic (ncp) BVDV can replicate in SSCs and that SSCs are capable of producing infectious BVDV. Giemsa staining showed significant changes in the morphology of SSCs after BVDV infection. Western blot and mRNA analysis showed that proliferation-related genes (PCNA, CCND1, CDK2) and SSC functional genes (Lin28A, OCT4, SOX2) were down regulated after infection. In addition, BVDV infection can induce ferroptosis in SSCs. Furthermore, CRISPR-Cas9 mediated editing of CD46 in goat SSCs resulted in a decrease in BVDV infection rate and alleviated the negative impact of the virus on cell survival and proliferation. This study provides new insights into the mechanism of reduced reproductive function in male livestock infected with BVDV, and lays the foundation for developing targeted disease resistant breeding strategies.},
}

Animals
Male
Goats
Cattle
CRISPR-Cas Systems
*Diarrhea Viruses, Bovine Viral/physiology/pathogenicity
*Bovine Virus Diarrhea-Mucosal Disease/virology
*Adult Germline Stem Cells/virology
Reproduction
Gene Knockout Techniques
*Spermatogonia/virology

@article {pmid41218271,
year = {2026},
author = {Fang, T and Peng, C and Ding, R and Fan, Y and Jia, J and Chen, J and Zhang, X and Wang, D and Li, J},
title = {Biomimetic nanoplatform-mediated CRISPR/Cas9 delivery for dual-pathway metabolic blockade in head and neck squamous cell carcinoma.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123837},
doi = {10.1016/j.biomaterials.2025.123837},
pmid = {41218271},
issn = {1878-5905},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Squamous Cell Carcinoma of Head and Neck/metabolism/drug therapy/pathology ; Cell Line, Tumor ; *Nanoparticles/chemistry ; *Head and Neck Neoplasms/metabolism/drug therapy/pathology ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Biomimetic Materials/chemistry ; Glycolysis/drug effects ; Mice ; Mice, Nude ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) continues to exhibit a poor prognosis, largely due to late diagnosis and the development of cisplatin resistance. Tumor proliferation in HNSCC is closely associated with upregulation of key glycolytic enzymes. However, monotherapeutic targeting of glycolysis paradoxically enhances compensatory glutaminolysis via glutamate overproduction. To overcome this metabolic adaptation, we developed biomimetic nanoparticles P-T-p@CM, fabricated from the pH/reduction dual-responsive copolymer poly(lactic acid)-polyhistidine-polyethylenimine (PLA-pHis-ss-PEI). This nanoplatform enables coordinated co-delivery of telaglenastat (a glutaminase 1 (GLS1) inhibitor) and a CRISPR-Cas9 plasmid encoding sgRNA targeting HIF-1α. This system utilizes homologous cancer cell membrane coating for precise tumor homing, with stimuli-responsive release enabling simultaneous dual metabolic blockade: CRISPR-mediated HIF-1α knockout attenuates glycolysis while telaglenastat suppresses glutamine-to-glutamate conversion. Metabolic analyses confirmed significant reduction in Glycolysis proton efflux rate (GlycoPER), the oxygen consumption rate (OCR) and ATP generation, as well as the related metabolites including the lactate production and glutamate. This dual-starvation strategy depleted energy reserves and biosynthetic precursors, inducing severe metabolic disruption. Notably, in vivo studies showed a 90 % tumor inhibition rate (TIR) after 15 days of treatment, through enhanced apoptosis, reduced proliferation, and tumor glucose/glutamate depletion. Collectively, P-T-p@CM establishes a paradigm-shifting approach to disrupt metabolic compensation in the treatment of HNSCC.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Squamous Cell Carcinoma of Head and Neck/metabolism/drug therapy/pathology
Cell Line, Tumor
*Nanoparticles/chemistry
*Head and Neck Neoplasms/metabolism/drug therapy/pathology
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
*Biomimetic Materials/chemistry
Glycolysis/drug effects
Mice
Mice, Nude

@article {pmid41213207,
year = {2026},
author = {Chen, J and Wang, Z and Du, Y and Liu, B and Chen, Y and Chen, G and Cui, W},
title = {Injectable CRISPRa-microspheres for targeted A20 activation rescue age-related osteogenic impairment via senescence mitigation.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123830},
doi = {10.1016/j.biomaterials.2025.123830},
pmid = {41213207},
issn = {1878-5905},
mesh = {Animals ; *Osteogenesis/genetics ; *Cellular Senescence/genetics ; Mice ; *Microspheres ; *Tumor Necrosis Factor alpha-Induced Protein 3/metabolism/genetics ; Mesenchymal Stem Cells/cytology/metabolism ; *Aging ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Gene Editing/methods ; Bone Regeneration ; Male ; Nanoparticles/chemistry ; Injections ; Humans ; },
abstract = {Age-related osteogenic failure in bone defect repair remains a significant clinical challenge, primarily due to persistent chronic inflammation-induced stem cell senescence. To address this, we engineered injectable CRISPRa-based gene-editing microspheres (GEMs), utilizing microfluidic-synthesized lipid nanoparticles (cLNPs) to co-deliver dCas9-VP64/sgRNA. This platform allows for precise spatiotemporal activation of tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) within bone marrow stromal cells (BMSCs), effectively reprogramming the senescence-osteogenesis axis. Our study identifies A20 as a key regulator of the senescence-associated secretory phenotype (SASP) and osteogenic impairment in aged BMSCs. In vitro, GEMs reduced senescence markers (p16 and p21) by over 30 %, while increasing osteogenic gene expression (RUNX2 and ALP) by 4 ∼ 5-fold, and suppressed inflammatory cytokines IL-6 and TNF-α by more than 30 %. In vivo, in aged mice with critical-sized bone defects, GEMs achieved a significant bone regeneration and promoted vascularization 3.1 times faster (CD31[+] staining) compared to controls. This GEM system offers a promising, clinically viable strategy for recalibrating age-related skeletal disorders, demonstrated by the precise targeting of host stem cells in situ and achieving approximately 80 % defect healing in aged bone defect models.},
}

Animals
*Osteogenesis/genetics
*Cellular Senescence/genetics
Mice
*Microspheres
*Tumor Necrosis Factor alpha-Induced Protein 3/metabolism/genetics
Mesenchymal Stem Cells/cytology/metabolism
*Aging
*CRISPR-Cas Systems/genetics
Mice, Inbred C57BL
Gene Editing/methods
Bone Regeneration
Male
Nanoparticles/chemistry
Injections
Humans

@article {pmid41187596,
year = {2026},
author = {Doherty-Boyd, WS and Tsimbouri, PM and Jayawarna, V and Walker, M and Taqi, AF and Mahon, N and Meek, D and Young, P and Miller, A and West, A and Salmeron-Sanchez, M and Dalby, MJ and Donnelly, H},
title = {Synthetic peptide hydrogels as a model of the bone marrow niche demonstrate efficacy of a combined CRISPR-CAR T-cell therapy for acute myeloid leukaemia.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123803},
doi = {10.1016/j.biomaterials.2025.123803},
pmid = {41187596},
issn = {1878-5905},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/pathology ; *Hydrogels/chemistry ; *Peptides/chemistry ; *CRISPR-Cas Systems ; *Bone Marrow/pathology ; Mesenchymal Stem Cells/cytology ; Gene Editing ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; *Immunotherapy, Adoptive/methods ; },
abstract = {Leukaemias, driven by mutations in haematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other haematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments' efficacy. Here, we present a bioengineered human-cell containing endosteal BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissue's mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.},
}

Humans
*Leukemia, Myeloid, Acute/therapy/pathology
*Hydrogels/chemistry
*Peptides/chemistry
*CRISPR-Cas Systems
*Bone Marrow/pathology
Mesenchymal Stem Cells/cytology
Gene Editing
*Receptors, Chimeric Antigen
T-Lymphocytes
*Immunotherapy, Adoptive/methods

@article {pmid41465590,
year = {2025},
author = {Nguyen, PD and Nakanishi, K and Nguyen, HP and Nguyen, HV and Kitao, M and Yoshimoto, M and Kamei, K},
title = {Characterisation of the Novel Cutibacterium acnes Phage KIT09 and First Report of CRISPR-Cas-Independent Bacteriophage Resistance in Phylotype IA1.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465590},
issn = {1422-0067},
support = {JPJSCCB20230005//Japan Society for the Promotion of Science/ ; },
mesh = {*Bacteriophages/genetics/isolation & purification/physiology ; *Propionibacterium acnes/virology/genetics ; *CRISPR-Cas Systems ; Humans ; Mutation ; Genome, Viral ; *Propionibacteriaceae/virology ; },
abstract = {Despite being a commensal bacterium, Cutibacterium acnes has been widely considered a major opportunistic pathogen due to its capacity for biofilm production and inflammatory induction, causing device-related, post-implant infections, and skin inflammatory diseases. In this study, we isolated and characterised the novel bacteriophage Cutibacterium acnes phage KIT09 as a potential antimicrobial candidate for the treatment of Cutibacterium acnes-related infections such as acne vulgaris and postsurgical infections. Subsequently, phage-resistant bacterial mutants were generated through phage KIT09 exposure and characterised. Wastewater samples were collected for the isolation of C. acnes phages, followed by their characterisation using C. acnes National Institute of Technology and Evaluation (NITE) Biological Resources Center (NBRC) 107605 (phylotype IA1). Resistant mutants were isolated after prolonged exposure of the newly isolated phage to host bacteria and then characterised. A novel C. acnes phage, designated KIT09, was isolated, demonstrating prolonged bacteriolysis lasting up to 96 h at a multiplicity of infection of 10, and exhibiting high thermal and pH stability. Following sustained selective pressure by phage KIT09, three phage-resistant bacterial isolates were obtained, forming smaller colonies than the wild-type strain, but maintaining a high phage adsorption capacity (>90% after 20 min). Whole-genome sequencing revealed 12 nucleotide mutations across five genes, including six non-synonymous substitutions. Three genes encoding a two-component histidine kinase, DNA processing protein A (DprA), and a ThuA-containing domain protein were mutated in all resistant isolates. Characterisation of the novel phage KIT09 demonstrated its robust lytic activity and environmental stability against C. acnes phylotype IA1. Isolated resistant mutants retained high phage adsorption, accompanied by recurrent mutations in genes encoding a two-component histidine kinase, DprA, and a ThuA-domain protein, suggesting the presence of alternative, CRISPR-Cas-independent resistance mechanisms in C. acnes.},
}

*Bacteriophages/genetics/isolation & purification/physiology
*Propionibacterium acnes/virology/genetics
*CRISPR-Cas Systems
Humans
Mutation
Genome, Viral
*Propionibacteriaceae/virology

@article {pmid41465565,
year = {2025},
author = {Mikhaylova, E and Khusnutdinov, E and Terekhov, M and Pozdeev, D and Gusev, O},
title = {Pig Genome Editing for Agriculture: Achievements and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465565},
issn = {1422-0067},
support = {075-15-2025-014 (075-15-2024-666)//The Ministry of Science and Higher Education of Russian Federation/ ; },
mesh = {*Gene Editing/methods ; Animals ; Swine/genetics ; *Agriculture/methods ; CRISPR-Cas Systems ; Breeding/methods ; *Genome ; },
abstract = {The remaining problems in pig farming may no longer be solved with traditional methods. The search for genetic variants associated with desired characteristics and involvement of animals with superior genetics in breeding programs is rarely effective for polygenic traits and pleiotropic genes. The lack of diversity in the germplasm also limits the use of breeding, but some beneficial mutations that did not occur naturally can be introduced manually via genome editing methods. Mutations discovered in other species, such as cattle, can be reproduced in pigs. Traits that were previously pursued for centuries might be achieved by genome editing in a few years. Enormous progress has been made in producing pigs resistant to viruses and in increasing meat productivity and quality. But there are still pressing problems such as lameness and damaging behaviors that probably cannot be solved without genome editing techniques. Their wider application is complicated by the requirement for large amounts of biomaterial, surgical manipulations and cell culture, as well as by the shift towards biomedical research. This review concentrates on the main achievements and challenges in pig agricultural genetics that can be addressed by genome editing.},
}

*Gene Editing/methods
Animals
Swine/genetics
*Agriculture/methods
CRISPR-Cas Systems
Breeding/methods
*Genome

@article {pmid41465342,
year = {2025},
author = {Sattarov, R and Kuznetsov, A and Klimko, V and Ignatyeva, E and Ivanov, R and Karabelsky, A and Fizikova, A},
title = {The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465342},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//"Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {*Hemophilia B/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Factor IX/genetics ; Humans ; CRISPR-Cas Systems ; Mutation ; Animals ; Genetic Vectors/genetics ; },
abstract = {Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.},
}

*Hemophilia B/therapy/genetics
*Genetic Therapy/methods
*Gene Editing/methods
*Factor IX/genetics
Humans
CRISPR-Cas Systems
Mutation
Animals
Genetic Vectors/genetics

@article {pmid41463573,
year = {2025},
author = {Li, Y and Gong, K and Wang, X and Sun, Z and Ding, F},
title = {Heat Shock Transcription Factors as Central Integrators of Plant Stress Responses: From Thermotolerance to Multi-Stress Resilience.},
journal = {Biology},
volume = {14},
number = {12},
pages = {},
pmid = {41463573},
issn = {2079-7737},
support = {ZR2025MS427//Shandong Provincial Natural Science Foundation/ ; 3247180862//National Natural Science Foundation of China/ ; },
abstract = {Heat shock transcription factors (HSFs) have long been recognized for their essential role in mediating thermotolerance via the activation of heat shock proteins (HSPs). Recent studies, however, have significantly broadened this view, revealing that HSFs function as versatile transcriptional regulators orchestrating plant adaptation to a wide range of abiotic and biotic stresses. This review synthesizes current knowledge of HSF structure, activation, and canonical roles in the heat shock response, while emphasizing emerging insights into their diverse functions beyond heat stress. Evidence from both model and crop species demonstrates that many HSFs confer tolerance to a broad range of stresses, including drought, cold, salinity, oxidative stress, and pathogen attack, through intricate crosstalk with hormonal (e.g., ABA, SA, JA) and redox signaling pathways, as well as MAPK-mediated phosphorylation. We also discuss biotechnological strategies such as CRISPR/Cas-mediated genome editing, stress-inducible promoter engineering, and synthetic transcriptional circuits that offer promising avenues for fine-tuning HSF expression and enhancing multi-stress resilience in crops. A deeper understanding of HSF multifunctionality not only advances our comprehension of plant stress biology but also provides a foundation for engineering resilient crops in the context of global climate change.},
}

@article {pmid41461563,
year = {2026},
author = {Riley, SE and Noskova Fairley, M and Xia, S and Cunningham, R and Cholewa-Waclaw, J and Feng, Y and Hansen, CG},
title = {In vivo screen reveals specific roles of Hippo pathway components in development and regeneration.},
journal = {Life science alliance},
volume = {9},
number = {3},
pages = {},
pmid = {41461563},
issn = {2575-1077},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Regeneration/genetics/physiology ; *Zebrafish Proteins/metabolism/genetics ; Signal Transduction/genetics ; Hippo Signaling Pathway ; *Protein Serine-Threonine Kinases/metabolism/genetics ; YAP-Signaling Proteins ; CRISPR-Cas Systems/genetics ; Macrophages/metabolism ; Larva ; Animal Fins/physiology ; Gene Expression Regulation, Developmental ; Transcription Factors/metabolism/genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; },
abstract = {The Hippo signalling pathway is a major regulator of regeneration and development. However, the comparative importance and functional roles of individual Hippo pathway components in vivo are greatly unknown, particularly within the vertebrate lineage. To gain direct and comparable insights, we took advantage of the zebrafish larva model system. We generated individual and combined CRISPR/Cas9 F0 knockouts of a range of core Hippo pathway genes, including upstream regulators, the co-transcriptional regulators Yap1/Taz, and Yap1/Taz target genes. We analysed and compared the resulting developmental and regenerative phenotypes. Our findings highlight that paralogues of core components have distinct, but in some instances overlapping, functions. Intriguingly, we find that Yap1 and Taz have differential roles during development and regeneration. In addition, we characterise and compare two tail fin regenerative paradigms: after both severe and mild injury. These injury paradigms are drastically different and elicit diverse resolution processes. We confirm critical roles of the immune system in the regenerative process. Macrophage recruitment is reduced during severe tail fin regeneration after Yap1 and Taz loss, appearing earlier in yap1 than wwtr1 Crispants and correlating with defective regenerative function. This defective macrophage involvement might therefore be one of the mediators of the deficient regeneration in these two Crispants. Overall, our analysis emphasises distinct requirements and responses of the Hippo pathway during development and across different regenerative paradigms.},
}

Animals
*Zebrafish/genetics/metabolism
*Regeneration/genetics/physiology
*Zebrafish Proteins/metabolism/genetics
Signal Transduction/genetics
Hippo Signaling Pathway
*Protein Serine-Threonine Kinases/metabolism/genetics
YAP-Signaling Proteins
CRISPR-Cas Systems/genetics
Macrophages/metabolism
Larva
Animal Fins/physiology
Gene Expression Regulation, Developmental
Transcription Factors/metabolism/genetics
Transcriptional Coactivator with PDZ-Binding Motif Proteins

@article {pmid41461407,
year = {2026},
author = {Shahid, M},
title = {Molecular engineering and in-silico biotechnological innovations for microbial degradation of persistent pesticides.},
journal = {Pesticide biochemistry and physiology},
volume = {217},
number = {},
pages = {106833},
doi = {10.1016/j.pestbp.2025.106833},
pmid = {41461407},
issn = {1095-9939},
mesh = {*Pesticides/metabolism ; Biodegradation, Environmental ; *Biotechnology ; Metabolic Engineering ; Soil Microbiology ; Bacteria/metabolism/genetics ; Computer Simulation ; },
abstract = {The persistence of recalcitrant pesticides in agricultural soils poses a serious threat to environmental and public health. Conventional remediation methods often have limited efficiency and, sustainability. Whereas, microbial degradation provides an eco-friendly and attractive alternative. This review highlights advances in molecular and biotechnological tools driving microbial pesticide degradation. It also emphasizes key genes, enzymatic pathways, and resilient microbes driving recalcitrant pesticide degradation. This review discusses the integration of next-generation sequencing, multi-omics platforms, CRISPR-Cas editing, synthetic biology, and AI-driven metabolic engineering in advancing microbial pesticide degradation. It also highlights progress in rhizosphere microbiome research, bioinformatics pipelines, and field-scale validation. The transition from lab to field highlights precision bioremediations' potential for sustainable pesticide management.},
}

*Pesticides/metabolism
Biodegradation, Environmental
*Biotechnology
Metabolic Engineering
Soil Microbiology
Bacteria/metabolism/genetics
Computer Simulation

@article {pmid41459216,
year = {2025},
author = {Sun, Y and Zhao, Q and Li, W and Kwok, LY and Zhang, H},
title = {Genomic diversity and functional adaptation of Limosilactobacillus reuteri isolated from diverse ecological niches.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1732127},
pmid = {41459216},
issn = {1664-302X},
abstract = {Limosilactobacillus reuteri is a widely utilized probiotic, however, the genomic diversity and evolutionary mechanisms underlying its adaptation to various hosts and environments remain incompletely understood. This study employed comparative genomics to analyze 176 L. reuteri genomes from animal (rodents, mammals, ruminants, and birds), human intestinal, and food sources (dairy products, fermented foods; 89 newly sequenced and 92 retrieved, 5 excluded by ANI < 95%). We assessed genomic features, average nucleotide identity, pan/core genomes, carbohydrate-active enzymes, bacteriocin production, CRISPR-Cas systems, and antibiotic resistance genes. The pan-genome consisted of 16,814 genes, while the core genome contained 553 genes. Core-gene phylogeny revealed seven clades, rodents isolates were positioned closer to the root. The clustering trend of fermented foods isolates in the phylogenetic tree may indicate that these strains have undergone convergent evolution or adaptive evolution in a specific environment. CAZymes varied across sources, and the predicted bacteriocin clusters were enriched in animal-derived, particularly in rodent isolates. CAZy functional composition in L. reuteri is shaped by the ecological niche and host environment, reflecting a pattern of host-driven evolutionary adaptation. CRISPR-Cas systems were present in 23.3% of genomes, predominantly in rodents isolates, indicating strong anti-phage capabilities. The heterogeneity of CRISPR-Cas systems among sources suggests that subpopulations of L. reuteri have been subjected to different evolutionary pressures. The predominance of Type II systems agrees with their widespread occurrence in lactobacilli. The presence of multiple probiotic function-related genes across all separation sources confirms the robust probiotic potential of L. reuteri. Antibiotic resistance genes, including tet, ermB, and vatE, were most prevalent among animal-derived isolates, with the highest numbers occurring in mammals and the lowest in rodents. Therefore, strain-specific safety assessments are necessary prior to clinical or food applications. The findings underscore the significance of host-specific adaptations in shaping the genetic and functional profiles of L. reuteri, offering valuable implications for its application in food-derived, human-derived, animal-derived and therapeutics.},
}

@article {pmid41458398,
year = {2025},
author = {Kang, J and Koo, J and Oh, H and Bae, E},
title = {Structural characterization of anti-CRISPR protein AcrIE9.},
journal = {Structural dynamics (Melville, N.Y.)},
volume = {12},
number = {6},
pages = {064701},
pmid = {41458398},
issn = {2329-7778},
abstract = {The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.},
}

@article {pmid41458320,
year = {2025},
author = {Ghiotto, G and Francescato, L and Biancalani, MA and Treu, L and Campanaro, S},
title = {Hydrogen excess drives metabolic reprogramming and viral dynamics in syngas-converting microbiomes.},
journal = {Environmental science and ecotechnology},
volume = {28},
number = {},
pages = {100637},
pmid = {41458320},
issn = {2666-4984},
abstract = {Microbial communities drive essential bioprocesses, including the conversion of synthesis gas into biomethane, a sustainable energy source that supports circular carbon economies. In anaerobic environments, specialized consortia of bacteria and archaea facilitate syngas methanation through syntrophic interactions, where hydrogenotrophic methanogens play a central role in reducing carbon dioxide and monoxide with hydrogen. However, imbalances in gas ratios, particularly excess hydrogen, can disrupt these interactions and impair overall efficiency. Yet, the molecular mechanisms underlying microbial responses to such imbalances remain poorly understood. Here we show that hydrogen excess triggers profound metabolic and viral remodeling in a thermophilic anaerobic microbiome, leading to reduced methane yields and ecological instability. This reprogramming involves transcriptional downregulation of methanogenesis genes in the dominant archaeon Methanothermobacter thermautotrophicus, coupled with upregulation of CRISPR-Cas and restriction-modification systems that correlate with diminished activity of an associated phage, indicating activated host defenses against viral threats. Concurrently, bacterial species such as those from Tepidanaerobacteraceae enhance carbon fixation via the Wood-Ljungdahl pathway, serving as electron sinks to mitigate redox imbalance. These adaptive responses highlight the microbiome's resilience mechanisms under stress, revealing viruses as both stressors and selective forces in syntrophic systems. Such insights advance our understanding of microbiome dynamics in bioconversion processes and guide the engineering of more stable microbial consortia for optimized syngas-to-methane conversion amid variable feedstocks.},
}

@article {pmid41457595,
year = {2025},
author = {Chang, Y and Fan, S and Hao, T and Dai, J and He, W},
title = {[Characterization of cbm2813 encoding the cytochrome P450 enzyme in the biosynthetic gene cluster of carbomycin].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {11},
pages = {4125-4137},
doi = {10.13345/j.cjb.250364},
pmid = {41457595},
issn = {1872-2075},
mesh = {*Multigene Family/genetics ; *Streptomyces/genetics/metabolism/enzymology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Macrolides/metabolism ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/biosynthesis ; },
abstract = {Carbomycin, a 16-membered macrolide antibiotic produced in Streptomyces thermotolerans, comprises two components, carbomycin A (CA) and carbomycin B (CB). CB is converted into CA through epoxidation of the C12-C13 double bond. The gene cbm2813, located in the biosynthetic gene cluster of carbomycin, encodes a cytochrome P450 enzyme considered to catalyze this epoxidation. In this study, the functional and enzymatic properties of the cytochrome P450 enzyme Cbm2813 in the carbomycin biosynthesis gene cluster were characterized by in vivo gene inactivation and in vitro enzymatic reactions. We employed the CRISPR-Cas9 system to delete cbm2813 and obtained the mutant Δcbm2813. The fermentation products of the mutant contained CB but not CA. Complementation of Δcbm2813 restored CA production. Cbm2813 was successfully expressed in Escherichia coli and then purified. In vitro enzyme assays confirmed that Cbm2813 specifically recognized CB but not structurally similar 16-membered macrolide antibiotics, such as josamycin, midecamycin, and isovalerylspiramycin I. Cbm2813 exhibited the maximal activity at pH 5.5 and 36 ℃, with the catalytic efficiency kcat/Km of 4.39×10[3] L/(mol·s). Molecular docking suggested that the C9 carbonyl group of CB coordinated with the heme iron in the active site of the enzyme, ensuring strict substrate specificity. This study expands the toolbox of characterized P450 enzymes and advances the understanding of carbomycin biosynthesis.},
}

*Multigene Family/genetics
*Streptomyces/genetics/metabolism/enzymology
*Cytochrome P-450 Enzyme System/genetics/metabolism
Escherichia coli/genetics/metabolism
Macrolides/metabolism
*Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems
*Anti-Bacterial Agents/biosynthesis

@article {pmid41457550,
year = {2025},
author = {Sun, Y and Li, W and Wang, P and Pei, J and Yang, L and Qiu, L and Liu, H},
title = {[CRISPRi-mediated regulation of nitrate metabolism genes in Chlamydomonas reinhardtii enhances lipid accumulation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4794-4809},
doi = {10.13345/j.cjb.250363},
pmid = {41457550},
issn = {1872-2075},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Lipid Metabolism/genetics ; *Nitrates/metabolism ; Nitrate Reductase/genetics/metabolism ; Nitrogen/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nitrite Reductases/genetics ; },
abstract = {The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference (CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene (CrNIT1) and the nitrite reductase gene (CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type (WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the CrNII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.},
}

*Chlamydomonas reinhardtii/genetics/metabolism
*Lipid Metabolism/genetics
*Nitrates/metabolism
Nitrate Reductase/genetics/metabolism
Nitrogen/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nitrite Reductases/genetics

@article {pmid41457547,
year = {2025},
author = {Song, S and Yao, D and Cai, Z and Yue, X and Qiao, C and Xue, C},
title = {[Construction of a CRISPR-Cas6-mediated lycopene synthase assembly regulation method].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4745-4758},
doi = {10.13345/j.cjb.250396},
pmid = {41457547},
issn = {1872-2075},
mesh = {Lycopene ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Intramolecular Lyases/genetics/metabolism ; Plasmids/genetics ; Carotenoids/metabolism ; },
abstract = {A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.},
}

Lycopene
*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Intramolecular Lyases/genetics/metabolism
Plasmids/genetics
Carotenoids/metabolism

@article {pmid41457318,
year = {2025},
author = {Brandt, D and Dörrich, AK and Persicke, M and Kemmler, A and Leonhard, T and Haak, M and Nölting, S and Ruwe, M and Schmid, N and Thormann, KM and Kalinowski, J},
title = {A pentose, as a cytosine nucleobase modification in Shewanella phage Thanatos genomic DNA, mediates enhanced resistance toward host restriction systems.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0133325},
doi = {10.1128/aem.01333-25},
pmid = {41457318},
issn = {1098-5336},
abstract = {UNLABELLED: Co-evolution of bacterial defense systems and phage counter-defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. In order to evade nuclease-based mechanisms that target DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits the recognition and restriction by endonucleases. We found that Shewanella phage Thanatos DNA is insensitive to multiple restriction enzymes and also to Cas I-Fv and Cas9 cleavage. Furthermore, with nanopore sequencing, the phage DNA showed severely impaired basecalling. In addition to an adenine methylation, the data indicated an additional, much more substantial nucleobase modification. Using liquid chromatography-mass spectrometry (LC-MS), we identified an unknown configuration of a deoxypentose attached to cytosine as an undiscovered modification of phage DNA, which is present in Thanatos genomic DNA and likely mediates resistance to restriction endonucleases, as well as reducing Cas nuclease activity significantly. To elucidate the underlying enzyme functions, we identified structural homologs of Thanatos proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of the phage protein TH1_063 by in vitro. Inactivation of TH1_060 leads to an almost complete inhibition of phage propagation, indicating an important role of the cytosine modification in phage survival and/or proliferation.

IMPORTANCE: Several phages extensively decorate their DNA building blocks, providing an effective protection against various host and phage-produced restriction systems. These modifications allow the phages to distinguish between their own DNA and that of the host, significantly increasing the establishment of the phage chromosome upon entry into the host and subsequent phage proliferation. Several different modifications have been previously identified and characterized. Here, we describe a hitherto unknown cytosine modification, consisting of a deoxypentose-putatively xylose-that provides protection against various bacterial restriction systems, including DNA-targeting CRISPR/Cas systems. Our findings expand the range of DNA modifications that phages use for protection.},
}

@article {pmid41403193,
year = {2025},
author = {Su, S and Zuo, Y and Ma, B and Zhao, Z and Wang, X and Zhang, X and Ignatus, AD and Piñero, JC and Peng, X and Li, F and Chen, M},
title = {Functional Validation of GmGSTs2 in the Resistance to Abamectin in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae).},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {52},
pages = {33033-33045},
doi = {10.1021/acs.jafc.5c12427},
pmid = {41403193},
issn = {1520-5118},
mesh = {Animals ; *Ivermectin/analogs & derivatives/pharmacology ; *Moths/genetics/drug effects/enzymology/growth & development ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance ; CRISPR-Cas Systems ; Larva/drug effects/genetics/growth & development/enzymology ; },
abstract = {Abamectin has been used for decades as an insecticide and acaricide in arthropod pest management. However, there is no direct evidence from CRISPR/Cas9 studies confirming the involvement of GSTs in insect resistance to abamectin. The oriental fruit moth, Grapholita molesta, is a destructive pest of fruit trees worldwide. The role of GSTs in the oriental fruit moth remains unclear. In this study, an abamectin-resistant strain (AB-R) was derived from a susceptible laboratory strain (AB-S) of G. molesta. Synergist bioassays showed that the GST inhibitor diethyl maleate (DEM) significantly increased abamectin toxicity in AB-R. Biochemical assays indicated that glutathione S-transferase (GST) activity in AB-R was 1.63-fold higher than in AB-S. Among 25 GST genes examined, GmGSTs2 showed the largest expression difference between AB-R and AB-S and was expressed across developmental stages and body parts. Recombinant GmGSTs2 significantly reduced the effective quantity of abamectin in vitro. CRISPR/Cas9 knockout of GmGSTs2 in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of G. molesta. The transgenic Drosophila melanogaster strain expressing GmGSTs2 showed an LC50 of 74.12 mg L[-1] (34.59-126.63) versus 25.48 mg L[-1] (12.28-39.82) in W[1118] controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, in vitro metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous in vivo assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in G. molesta, providing a practical target for resistance management.},
}

Animals
*Ivermectin/analogs & derivatives/pharmacology
*Moths/genetics/drug effects/enzymology/growth & development
*Insecticides/pharmacology
*Glutathione Transferase/genetics/metabolism
*Insect Proteins/genetics/metabolism
Insecticide Resistance
CRISPR-Cas Systems
Larva/drug effects/genetics/growth & development/enzymology

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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