@article {pmid41191093,
year = {2025},
author = {Sun, Y and Han, H and Zhao, R and Liu, X and Liu, Z and Liang, J and Wang, B and Zhao, B and Pang, Y and Xiao, R},
title = {CRISPR-assisted MoS2@TQD nanosheets-based lateral flow assay for dual-mode and sensitive diagnosis of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {792},
pmid = {41191093},
issn = {1436-5073},
mesh = {Humans ; *SARS-CoV-2/isolation & purification/genetics ; *COVID-19/diagnosis ; *Molybdenum/chemistry ; *Quantum Dots/chemistry ; Limit of Detection ; *Disulfides/chemistry ; Colorimetry/methods ; CRISPR-Cas Systems ; Nanostructures/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; COVID-19 Testing/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) assembled lateral flow assay (LFA) have been developed for rapid virus diagnosis, however, the single-mode visual half-quantitative characteristics limit their applications in accurate quantitative diagnosis. Herein, a CRISPR assisted MoS2@TQD (MoS2 nanosheet assembled ternary quantum dot) based colorimetric and fluorescent dual-mode LFA was fabricated for the ultrasensitive and flexible detection of the SARS-CoV-2. Compared with single QDs, MoS2@TQD can provide stable and superior colorimetric/fluorescent signals in various bio-samples. RT-RAA/CRISPR-Cas13 was adopted to couple with MoS2@TQD so that the detection sensitivity can be further enhanced. Based on the dual-mode LFA, the colorimetric visual detection limit was 400 copies/mL, and for fluorescence quantification mode, as low as 250 copies/mL can be obtained. For clinic diagnosis, 35 SARS-CoV-2 positive throat swab samples and 18 negative samples were detected by this LFA, 100% accuracy and 100% consistency with quantitative polymerase chain reaction (qPCR) results can be obtained. It indicated that MoS2@TQD-based dual-mode LFA can be a rapid, sensitive, and accurate detection platform for SARS-CoV-2 and other infectious pathogens in the point-of-care diagnosis.},
}

Humans
*SARS-CoV-2/isolation & purification/genetics
*COVID-19/diagnosis
*Molybdenum/chemistry
*Quantum Dots/chemistry
Limit of Detection
*Disulfides/chemistry
Colorimetry/methods
CRISPR-Cas Systems
Nanostructures/chemistry
*Clustered Regularly Interspaced Short Palindromic Repeats
COVID-19 Testing/methods

@article {pmid41188812,
year = {2025},
author = {Hussain, T and Awasthi, S and Shahid, F and Yi, SS and Sahni, N and Aldaz, CM},
title = {Therapeutic potential of PRMT1 as a critical survival dependency target in multiple myeloma.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1704},
pmid = {41188812},
issn = {1471-2407},
support = {R35GM133658/GM/NIGMS NIH HHS/United States ; R35GM137836/NH/NIH HHS/United States ; 7016-18//Leukemia and Lymphoma Society/ ; },
mesh = {*Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics/metabolism ; Humans ; *Multiple Myeloma/drug therapy/genetics/pathology/metabolism ; Cell Survival/drug effects ; Cell Line, Tumor ; *Repressor Proteins/antagonists & inhibitors/genetics/metabolism ; Arginine/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; DNA Damage ; },
abstract = {Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.},
}

*Protein-Arginine N-Methyltransferases/antagonists & inhibitors/genetics/metabolism
Humans
*Multiple Myeloma/drug therapy/genetics/pathology/metabolism
Cell Survival/drug effects
Cell Line, Tumor
*Repressor Proteins/antagonists & inhibitors/genetics/metabolism
Arginine/metabolism
Gene Expression Regulation, Neoplastic/drug effects
CRISPR-Cas Systems
Cell Cycle/drug effects
DNA Damage

@article {pmid41188277,
year = {2025},
author = {Yeh, TY and Chiu, IM and Huang, YJ and Wu, SC and Hsieh, CC and Sung, HH and Hwu, WL and Wang, GJ and Ju, JC},
title = {Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {38648},
pmid = {41188277},
issn = {2045-2322},
support = {108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Blastocyst/metabolism/cytology ; *Kidney/metabolism/embryology ; Mice, Knockout ; Female ; Gene Targeting ; RNA, Guide, CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.},
}

Animals
*CRISPR-Cas Systems
Mice
*Blastocyst/metabolism/cytology
*Kidney/metabolism/embryology
Mice, Knockout
Female
Gene Targeting
RNA, Guide, CRISPR-Cas Systems/genetics
Green Fluorescent Proteins/genetics/metabolism
Gene Editing

@article {pmid41187082,
year = {2025},
author = {Avaro, AS and Mirjalili, S and Griffiths, AD and Santiago, JG},
title = {Microfluidic networks using isotachophoresis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {45},
pages = {e2511724122},
doi = {10.1073/pnas.2511724122},
pmid = {41187082},
issn = {1091-6490},
support = {//Swedish e-Science Research Centre (SeRC)/ ; //Kungliga Tekniska Högskolan (KTH)/ ; },
abstract = {The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.},
}

@article {pmid41171899,
year = {2025},
author = {Gil-Campillo, C and Mignolet, J and Domínguez-San Pedro, A and Rapún-Araiz, B and Janssen, AB and de Bakker, V and Veening, JW and Garmendia, J},
title = {CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants.},
journal = {PLoS pathogens},
volume = {21},
number = {10},
pages = {e1013650},
pmid = {41171899},
issn = {1553-7374},
mesh = {*Haemophilus influenzae/genetics/growth & development ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Genomics/methods ; },
abstract = {Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and we refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.},
}

*Haemophilus influenzae/genetics/growth & development
*Genome, Bacterial
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation, Bacterial
Genomics/methods

@article {pmid41109141,
year = {2026},
author = {Song, L and Bu, S and Ding, Y and Chai, Y and Fu, Y and Zhang, P and Yuan, R},
title = {CRISPR-enhanced low-potential ECL sensor based on AuPt-functionalized porphyrin AlOG for ultrasensitive aflatoxin B1 detection.},
journal = {Biosensors & bioelectronics},
volume = {292},
number = {},
pages = {118089},
doi = {10.1016/j.bios.2025.118089},
pmid = {41109141},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/isolation & purification/chemistry ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *Porphyrins/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Aluminum/chemistry ; Food Contamination/analysis ; },
abstract = {Herein, an ultrasensitive low-potential electrochemiluminescence (ECL) sensor was developed by integrating an AuPt nanoparticle-functionalized porphyrin aluminum-organic gel (P-AlOG@AuPt) emitter with an enhanced CRISPR/Cas12a amplification module for the detection of aflatoxin B1 (AFB1) related to food safety. Impressively, the P-AlOG@AuPt emitter exhibited strong ECL emission and high signal-to-noise ratio at a low triggering potential (-0.8 V), addressing key limitations of previous high-potential aluminum-organic gels. Meanwhile, the in situ enriched AuPt as a coreaction accelerator catalyzed the reduction of coreactant S2O8[2-] to produce abundant SO4[•-] for facilitating its interactions with P-AlOG@AuPt[•-], resulting in a significant ECL enhancement. Furthermore, a synergistic catalytic hairpin assembly (CHA) pre-amplification and tetrahedral DNA nanostructure (TDN)-anchored ssDNA reporters significantly improved the cleavage efficiency of CRISPR/Cas12a, boosting the detection sensitivity of the ECL sensor for AFB1. The established ECL sensor exhibited ultrasensitive detection of AFB1 with a linear range of 0.1 pg mL[-1] - 500 ng mL[-1] and detection limit of 0.044 pg mL[-1], outperforming most reported ECL- and CRISPR-based AFB1 sensors. The practical usability of the proposed ECL sensor was verified by applying it for the quantification of AFB1 in rice samples. This work offers a promising low-potential ECL sensing platform for practical mycotoxin monitoring and broader biosensing applications.},
}

*Aflatoxin B1/analysis/isolation & purification/chemistry
*Biosensing Techniques/methods
CRISPR-Cas Systems
Limit of Detection
Luminescent Measurements/methods
Electrochemical Techniques/methods
*Porphyrins/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
Aluminum/chemistry
Food Contamination/analysis

@article {pmid41052497,
year = {2026},
author = {Huang, X and Wang, A and Lin, Z and Xu, Y and Zheng, J},
title = {Novel electrochemiluminescence resonance energy transfer biosensor driven by CRISPR-Cas12a system for ctDNA detection.},
journal = {Biosensors & bioelectronics},
volume = {292},
number = {},
pages = {118067},
doi = {10.1016/j.bios.2025.118067},
pmid = {41052497},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Circulating Tumor DNA/genetics/blood/isolation & purification ; *Lung Neoplasms/genetics/blood/diagnosis ; *Luminescent Measurements/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Gold/chemistry ; *Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis ; Energy Transfer ; Mutation ; },
abstract = {Efficient energy donor-acceptor pairing and distance control are critical in resonance energy transfer-based electrochemiluminescence (ECL-RET) biosensor. In this work, a novel ECL-RET system with Ru(phen)3[2+] as ECL energy donor and Au nanocages as energy acceptor was constructed based on the characteristic that Ru(phen)3[2+] can be stably embedded into double-stranded DNA. On this basis, a homogeneous ECL biosensor based on target activated CRISPR-Cas12a system and driving ECL-RET effect was developed. The ECL biosensor has been successfully applied to the detection of circulating tumor DNA (ctDNA) and demonstrated efficient recognition of L858R mutation in ctDNA of non-small cell lung cancer patients. The presented ECL-RET biosensor achieves a wide linear detection range from 10 fM to 1 nM and an excellent detection limit as low as 3.0 fM for ctDNA due to its combination of the highly efficient and specific cleavage of the CRISPR-Cas12a system.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Circulating Tumor DNA/genetics/blood/isolation & purification
*Lung Neoplasms/genetics/blood/diagnosis
*Luminescent Measurements/methods
Limit of Detection
Electrochemical Techniques/methods
Gold/chemistry
*Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis
Energy Transfer
Mutation

@article {pmid40883986,
year = {2025},
author = {Jenny Gao, and Bamidele, N and Pires-Ferreira, D and Zheng, C and Destefano, A and Cheng, H and Tang, Q and Cao, Y and Xie, J and Gao, G and Gruntman, A and Sontheimer, E and Flotte, TR and Xue, W},
title = {A compact base editor rescues AATD-associated liver and lung disease in mouse models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5817-5828},
pmid = {40883986},
issn = {1525-0024},
support = {UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01 CA275945/CA/NCI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R01 GM150273/GM/NIGMS NIH HHS/United States ; F30 HL176024/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing/methods ; *alpha 1-Antitrypsin/genetics ; *alpha 1-Antitrypsin Deficiency/genetics/therapy/complications ; *Liver Diseases/therapy/genetics/etiology ; *Lung Diseases/therapy/genetics/etiology ; Mice, Transgenic ; Humans ; *Genetic Therapy/methods ; Mutation ; CRISPR-Cas Systems ; Dependovirus/genetics ; Genetic Vectors/genetics ; },
abstract = {Alpha-1 antitrypsin deficiency (AATD) is commonly caused by a G-to-A mutation in the SERPINA1 gene (the PiZ mutation). The mutant PiZ AAT protein is sequestered in hepatocytes, causing lung emphysema due to insufficient AAT protein to inhibit neutrophil elastase in the lung. Here, we show that a compact adenine base editor (ABE) with an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2.C) can be packaged in a single AAV and correct the PiZ mutation in mouse models of AATD. An all-in-one eNme2.C-TadA8e/guide 2 plasmid achieved approximately 20% on-target editing in PiZ reporter cells. TadA9e demonstrated sequence-specific reduction in bystander editing compared to TadA8e, without significantly affecting the on-target edit. In PiZ transgenic mice, eNme2.C-TadA9e AAV showed approximately 23% editing efficiency after 8 weeks and reduced liver disease burden in treated mice. In a new AAT-null;PiZ transgenic mouse model, ABE restored serum levels of AAT to beyond the 570 μg/mL therapeutic level. Moreover, ABE treatment was able to significantly correct lung functions in AAT-null;PiZ animals with emphysema. This study demonstrates the feasibility of an eNme2.C-based ABE in a single AAV to treat both AATD-associated liver and lung disease.},
}

Animals
Mice
Disease Models, Animal
*Gene Editing/methods
*alpha 1-Antitrypsin/genetics
*alpha 1-Antitrypsin Deficiency/genetics/therapy/complications
*Liver Diseases/therapy/genetics/etiology
*Lung Diseases/therapy/genetics/etiology
Mice, Transgenic
Humans
*Genetic Therapy/methods
Mutation
CRISPR-Cas Systems
Dependovirus/genetics
Genetic Vectors/genetics

@article {pmid40842155,
year = {2025},
author = {Ibel, A and Bhardwaj, R and Yilmaz, DE and Kong, S and Wendlinger, S and Cordero, C and Papaioannou, D and Papazian, M and Schönauer, R and Meng, Q and Eckardt, KU and Hassan, F and Volpe, I and Klämbt, V and Halbritter, J and Fedeles, S and Krappitz, M and Kaminski, MM},
title = {In vivo base editing reduces liver cysts in autosomal dominant polycystic kidney disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5373-5382},
doi = {10.1016/j.ymthe.2025.08.026},
pmid = {40842155},
issn = {1525-0024},
mesh = {*Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; Animals ; *Gene Editing/methods ; Mice ; Humans ; TRPP Cation Channels/genetics ; *Cysts/genetics/therapy/pathology/etiology ; *Liver Diseases/genetics/therapy/etiology/pathology ; Disease Models, Animal ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Liver/pathology/metabolism ; Mutation, Missense ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants in PKD1 or PKD2 account for 85%-90% of cases. Genetic re-expression of Pkd1 or Pkd2 has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in understanding the genetic basis, it remains unclear whether correcting pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of genome editing as a treatment remains largely unexplored. In this study, we employed CRISPR base editing to correct representative pathogenic PKD1 variants selected from a patient cohort, achieving precise and efficient editing in vitro. Correction of a murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells increased polycystin-1 expression and reduced the endoplasmic reticulum stress marker sXBP1. In vivo, base editor delivery to the c.6646C>T (R2216W) knockin mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence that genome editing may ameliorate key features of ADPKD, opening promising therapeutic perspectives for affected patients and their families.},
}

*Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology
Animals
*Gene Editing/methods
Mice
Humans
TRPP Cation Channels/genetics
*Cysts/genetics/therapy/pathology/etiology
*Liver Diseases/genetics/therapy/etiology/pathology
Disease Models, Animal
CRISPR-Cas Systems
Genetic Therapy/methods
Liver/pathology/metabolism
Mutation, Missense

@article {pmid40833860,
year = {2025},
author = {Yazdani, R and Nasr Esfahani, MH and Eghbalsaied, S and Karamali, F},
title = {Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla.},
journal = {DNA and cell biology},
volume = {44},
number = {11},
pages = {618-627},
doi = {10.1177/10445498251370554},
pmid = {40833860},
issn = {1557-7430},
mesh = {*CRISPR-Cas Systems/genetics ; *Liposomes/chemistry ; *Nanoparticles/chemistry ; *Extracellular Vesicles/metabolism/chemistry ; *Hepatocyte Growth Factor/genetics ; Humans ; *Stem Cells/metabolism ; Gene Editing/methods ; },
abstract = {Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.},
}

*CRISPR-Cas Systems/genetics
*Liposomes/chemistry
*Nanoparticles/chemistry
*Extracellular Vesicles/metabolism/chemistry
*Hepatocyte Growth Factor/genetics
Humans
*Stem Cells/metabolism
Gene Editing/methods

@article {pmid40808259,
year = {2025},
author = {Mamia, K and Kolbeinsdottir, S and Labun, K and Li, Z and Komisarczuk, A and Keskitalo, S and Reint, G and Loe Haugen, F and Olaug Lindestad, B and Skundberg Jensen, S and Gjerdingen, TJ and Tuhkala, A and Wieczorek Ervik, C and Kopcil, P and Fatkhutdinov, N and Bronken Martinsen, KH and Erichsen, HC and Szymanska, M and Tölö, E and Glumoff, V and Saarela, J and Melbye Michelsen, T and Schalin-Jäntti, C and Olweus, J and Leinonen, E and Varjosalo, M and Valen, E and Hautala, T and Enge, M and Martelius, T and Dahal-Koirala, S and Haapaniemi, E},
title = {Precision T cell correction platform for inborn errors of immunity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5624-5643},
doi = {10.1016/j.ymthe.2025.08.018},
pmid = {40808259},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; *T-Lymphocytes/metabolism/immunology ; Humans ; CRISPR-Cas Systems ; Animals ; Mice ; STAT1 Transcription Factor/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knockin, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation, or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single-nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function, cartilage hair hypoplasia, deficiency of ADA2, and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.},
}

*Gene Editing/methods
*T-Lymphocytes/metabolism/immunology
Humans
CRISPR-Cas Systems
Animals
Mice
STAT1 Transcription Factor/genetics
Genetic Therapy/methods
Disease Models, Animal

@article {pmid40739756,
year = {2025},
author = {Skov, TW and Wolff, JH and Haslund, D and Revenfeld, AL and van de Venn, L and Dorset, SR and Ravendran, S and Jørgensen, SE and Thomsen, MK and Holm, M and Corn, JE and Møller, BK and Mogensen, TH and Bak, RO and Mikkelsen, JG},
title = {Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5644-5660},
doi = {10.1016/j.ymthe.2025.07.038},
pmid = {40739756},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism ; Humans ; Alleles ; *Gene Editing/methods ; *GATA2 Transcription Factor/genetics/deficiency ; *GATA2 Deficiency/therapy/genetics ; Dependovirus/genetics ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Mice ; Genetic Vectors/genetics ; Recombinational DNA Repair ; },
abstract = {GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.},
}

*CRISPR-Cas Systems
*Hematopoietic Stem Cells/metabolism
Humans
Alleles
*Gene Editing/methods
*GATA2 Transcription Factor/genetics/deficiency
*GATA2 Deficiency/therapy/genetics
Dependovirus/genetics
*Genetic Therapy/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Mice
Genetic Vectors/genetics
Recombinational DNA Repair

@article {pmid40652526,
year = {2025},
author = {Oksenych, V and Petakh, P and Kainov, D and Kamyshnyi, O},
title = {A CRISPR-Cas9-based tool for dose-dependent DNA damage detection.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5625-5628},
doi = {10.1111/febs.70186},
pmid = {40652526},
issn = {1742-4658},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *DNA Damage/genetics ; Retroelements/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins ; },
abstract = {Auboiron et al. developed a CRISPR-Cas9-based system in yeast Saccharomyces cerevisiae that allows precise, dose-dependent induction of DNA double-strand breaks by targeting Ty retrotransposons. This system combines sequence specificity with control over break numbers (×1, ×15, or ×59), enabling a more detailed and precise study of DNA damage response. It revealed that the key DNA damage response kinase, Tel1, localizes to the nuclear periphery and forms multiple foci after induction of DNA double-strand breaks. The system also exposed limitations in Cas9 availability at higher break levels and offers a scalable platform for studying genome stability across organisms.},
}

*CRISPR-Cas Systems/genetics
*Saccharomyces cerevisiae/genetics
Saccharomyces cerevisiae Proteins/genetics/metabolism
*DNA Breaks, Double-Stranded
*DNA Damage/genetics
Retroelements/genetics
Protein Serine-Threonine Kinases/genetics/metabolism
Intracellular Signaling Peptides and Proteins

@article {pmid40631397,
year = {2025},
author = {Auboiron, M and Coiffard, J and Kumanski, S and Santt, O and Pardo, B and Moriel-Carretero, M},
title = {A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand break sensing and repair.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5629-5658},
doi = {10.1111/febs.70143},
pmid = {40631397},
issn = {1742-4658},
support = {Laureate 2018//ATIP-Avenir Program/ ; ANR-21-CE12-0004-01//Agence Nationale de la Recherche/ ; ARCPJA22020060002119//Fondation ARC pour la Recherche sur le Cancer/ ; PLBIO19-098 INCA_13832//Institut National Du Cancer/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; },
abstract = {The integrity of DNA is put at risk by different lesions, among which double-strand breaks (DSBs) occur at a lower frequency but have the most life-threatening consequences. The study of DSB repair requires tools that can induce the accumulation of these breaks and includes the use of chemical genotoxins, ionizing radiation, or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have leveraged the repetitive nature of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15, or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensin when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.},
}

*DNA Breaks, Double-Stranded
*Saccharomyces cerevisiae/genetics/metabolism
*Saccharomyces cerevisiae Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*DNA Repair/genetics
Protein Serine-Threonine Kinases/genetics/metabolism
Intracellular Signaling Peptides and Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics

@article {pmid40501348,
year = {2025},
author = {Rafiq, S and Assad, A},
title = {RNAS-sgRNA: Recurrent Neural Architecture Search for Detection of On-Target Effects in Single Guide RNA.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {32},
number = {11},
pages = {1041-1059},
doi = {10.1089/cmb.2025.0031},
pmid = {40501348},
issn = {1557-8666},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neural Networks, Computer ; Computational Biology/methods ; Algorithms ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a leading genomic editing tool, but its effectiveness is limited by considerable heterogeneity in target efficiency among different single guide RNAs (sgRNA). This study presents RNAS-sgRNA, a hybrid model that integrates neural architecture search (NAS) with recurrent neural networks (RNN) to evaluate the on-target efficacy of CRISPR/Cas9 sgRNA. The RNAS-sgRNA model automates architectural discovery, improving sgRNA sequence categorization without considerable manual adjustment. The NAS component improves the RNN architecture, which analyzes sgRNA sequences represented as binary matrices and produces a classification score. Upon evaluation across several datasets, RNAS-sgRNA exhibits substantial performance enhancements with multiple cell lines, comparing its area under the receiver operating characteristic curve (AUROC) performance to the baseline CRISPRpred(SEQ) and DeepCRISPR models. RNAS-sgRNA demonstrated substantial improvements in AUROC performance in several cell lines compared with existing models. Notable improvements include enhancements of 8.62% for HCT116, 121.57% for HEK293T, 13.40% for HeLa, and 20.78% for HL60 cell lines, resulting in an overall improvement of 13.46%. Compared with DeepCRISPR, the model achieved additional AUROC gains in all cell lines tested, with an average improvement of 14.74%. The study also highlighted the ability of the model to deliver superior performance on smaller datasets through transfer learning, underscoring its potential applications in personalized medicine and genetic research. RNAS-sgRNA introduces a novel integration of NAS with RNN to evaluate the efficacy of CRISPR/Cas9 sgRNA. Unlike traditional methods that require significant manual adjustments, this model automates architectural discovery, optimizing the RNN structure for sgRNA sequence analysis. Furthermore, the application of transfer learning to fine-tune the pretrained model on small cell-line datasets represents a pioneering approach in the domain. The model's demonstrated ability to significantly outperform existing algorithms, including CRISPRpred(SEQ) and DeepCRISPR, across multiple cell lines highlights its innovative contribution to genomic editing research and personalized medicine.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Neural Networks, Computer
Computational Biology/methods
Algorithms

@article {pmid41184760,
year = {2025},
author = {Guo, F and Wang, D and Wu, H and Zhao, X and Xu, F and Cui, Y and Shao, Y and Zhang, Y and Bu, W and Wen, T},
title = {Genomic characteristics, antimicrobial resistance profiles and virulence factors of Gallibacterium anatis isolates from layer chickens in Northern China.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {706},
pmid = {41184760},
issn = {1471-2180},
support = {31802193//National Natural Science Foundation of China/ ; XMS202505//the R & D Foundation of Institute of Animal Husbandry and Veterinary Medicine/ ; KJCX20230427//the Special Program on Science and Technology Innovation Capacity Building of BAAFS/ ; },
mesh = {Animals ; *Chickens/microbiology ; China ; *Virulence Factors/genetics ; *Poultry Diseases/microbiology ; Phylogeny ; *Pasteurellaceae/genetics/drug effects/pathogenicity/isolation & purification/classification ; *Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Pasteurellaceae Infections/veterinary/microbiology ; Drug Resistance, Multiple, Bacterial/genetics ; Virulence ; Genomics ; *Drug Resistance, Bacterial ; },
abstract = {BACKGROUND: Gallibacterium anatis is an emerging pathogen causing substantial economic losses in global poultry production. Despite its growing clinical significance, the genomic basis of pathogenicity and antimicrobial resistance in this species remains poorly understood, particularly in China.

RESULTS: We sequenced and analyzed five clinical G. anatis isolates from Chinese layer chickens alongside 31 global strains, their genomes ranged from 2.25 to 2.81 Mb with 39.8% average GC content. Phylogenetic analysis revealed that Chinese isolates cluster according to historical breeding stock importation patterns, reflecting international trade influences on pathogen distribution. Contemporary isolates showed extensive multidrug resistance compared to the antimicrobial-sensitive historical strain F149, with resistance profiles correlating directly with documented antibiotic usage in Chinese poultry production. Virulence analysis identified universal conservation of the RTX toxin system (97-100% prevalence) across all strains, contrasting sharply with variable distribution of other factors including fimbriae (30-35%) and secretion systems (50-60%). All strains harbored CRISPR-Cas systems, predominantly types I and III, indicating strain-specific phage defense adaptations.

CONCLUSIONS: The RTX toxin system represents a core virulence mechanism and potential universal vaccine target for G. anatis. The rapid evolution from antimicrobial-sensitive to extensively resistant phenotypes demonstrates how global poultry trade accelerates both pathogen spread and resistance development. These findings provide molecular insights for targeted interventions against an increasingly problematic poultry pathogen.},
}

Animals
*Chickens/microbiology
China
*Virulence Factors/genetics
*Poultry Diseases/microbiology
Phylogeny
*Pasteurellaceae/genetics/drug effects/pathogenicity/isolation & purification/classification
*Genome, Bacterial
Anti-Bacterial Agents/pharmacology
*Pasteurellaceae Infections/veterinary/microbiology
Drug Resistance, Multiple, Bacterial/genetics
Virulence
Genomics
*Drug Resistance, Bacterial

@article {pmid41184307,
year = {2025},
author = {White, N and Chalk, JA and Hu, YT and Pins, SM and Joseph, CR and Antoniou, P and Wimberger, S and Svensson, S and Caetano-Silva, SP and Mudde, ACA and Rai, R and Selvaraj, S and Feist, WN and Romito, M and Sienski, G and Nitsch, R and Booth, C and Santilli, G and Cavazza, A and Porteus, MH and Maresca, M and Thrasher, AJ and Turchiano, G},
title = {Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9571},
pmid = {41184307},
issn = {2041-1723},
support = {217112/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *T-Lymphocytes/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; *Endonucleases/metabolism/genetics ; Mutation ; },
abstract = {DNA repair mechanisms in human primary cells, including error-free repair, and, recurrent nuclease cleavage events, remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR), an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs, small indels, large deletions, and other aberrations in absolute terms in clinically relevant edited cells, including HSPCs, iPSCs, and T-cells. By quantifying up to 90% of loci with unresolved DSBs, CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore, we accurately quantify DNA repair precision, revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics, paving the way for mechanistic studies to advance gene therapy, designer editors, and small molecule discovery.},
}

Humans
*DNA Repair/genetics
*T-Lymphocytes/metabolism
Gene Editing/methods
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Induced Pluripotent Stem Cells/metabolism
*Endonucleases/metabolism/genetics
Mutation

@article {pmid41184277,
year = {2025},
author = {Nguyen, C and Omotayo, AI and Sanz Juste, S and Feng, X and López Del Amo, V},
title = {A temperature-sensitive CRISPR-Cas12a system for sterile insect technique.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9677},
pmid = {41184277},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Male ; Female ; Temperature ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; *Infertility, Male/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.},
}

Animals
*CRISPR-Cas Systems/genetics
Male
Female
Temperature
RNA, Guide, CRISPR-Cas Systems/genetics
Animals, Genetically Modified
*Infertility, Male/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
Gene Editing/methods
*Bacterial Proteins/genetics/metabolism

@article {pmid41106454,
year = {2025},
author = {Xie, D and Wang, J and Huang, L and Zhao, Z and Wang, R and Long, H and Ni, Q and Liu, H and Hu, M and Huang, J},
title = {A novel X-linked retinitis pigmentosa beagle dog model for ophthalmic research established rapidly by editing the RPGR gene in zygotes.},
journal = {Experimental eye research},
volume = {261},
number = {},
pages = {110697},
doi = {10.1016/j.exer.2025.110697},
pmid = {41106454},
issn = {1096-0007},
mesh = {Animals ; *Retinitis Pigmentosa/genetics/metabolism/physiopathology ; Dogs ; *Disease Models, Animal ; *Eye Proteins/genetics ; *Gene Editing/methods ; *Genetic Diseases, X-Linked/genetics ; Female ; *Zygote/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Male ; Mutation ; },
abstract = {Inherited retinal diseases (IRDs) constitute several ocular disorders leading to progressive and severe visual impairment. While significant progress has been made in understanding the genetic basis of IRDs, preclinical animal models are crucial for advancing therapeutic development. Although well-established mouse models exist, the scarcity of large animal models represents a significant limitation. Dogs (Canis familiaris) exhibit numerous physiological and anatomical similarities to humans, rendering them as potential models. X-linked retinitis pigmentosa (XLRP) is a severe, early-onset form of IRDs, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Here, we efficiently generated a stably inherited RPGR-knockout beagle model of XLRP in one-step via CRISPR/Cas9 gene editing tool with multiple gRNAs and zygote microinjection technology. Two F0 generation beagle dogs were successfully obtained with high knockout efficiency in the RPGR gene and the F0 female exhibited characteristic XLRP phenotypes, including progressive deterioration of both rod and cone photoreceptor function, a gradual reduction in the outer nuclear layer thickness from 7 to 35 months, and attenuated retinal arteries with minimal pigmentation. Notably, the F1 male of hemizygous RPGR-knockout beagle dogs displayed more pronounced disease phenotypes by 7 months of age. In conclusion, we successfully generated a stably inherited beagle dog model of XLRP exhibiting an intermediate characteristic disease onset, which is highly suitable for therapeutic research in gene therapy and stem cell transplantation for determining optimal intervention timing and therapeutic dosing.},
}

Animals
*Retinitis Pigmentosa/genetics/metabolism/physiopathology
Dogs
*Disease Models, Animal
*Eye Proteins/genetics
*Gene Editing/methods
*Genetic Diseases, X-Linked/genetics
Female
*Zygote/metabolism
Electroretinography
CRISPR-Cas Systems
Male
Mutation

@article {pmid41081521,
year = {2025},
author = {Wu, H and Lin, X and Tian, C and Xiong, D},
title = {Establishment of an RAA-CRISPR/Cas12a assay based on CpSge1 for rapid detection of Cryphonectria parasitica.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0107925},
pmid = {41081521},
issn = {2165-0497},
support = {2023YFD1401301//National Key Research and Development Program/ ; },
mesh = {*Ascomycota/genetics/isolation & purification ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Fagaceae/microbiology ; Fungal Proteins/genetics ; Recombinases/metabolism/genetics ; China ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Chestnut blight disease caused by Cryphonectria parasitica is a serious branch disease that occurs worldwide, especially in Europe and North America. In recent years, chestnut blight disease has also been severe and even posed a great threat to the healthy development of chestnut orchards in some areas of China. Accurate and rapid detection of C. parasitica during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the CpSge1 (Gti1/Pac2 transcription factor family) of C. parasitica as the detection target and established a rapid and visual detection system of C. parasitica that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called CpSge1-RAA-CRISPR/Cas12a. The system allows for the specific detection of C. parasitica in approximately 60 mins, with visualization of results. The detection sensitivity of this system was found to be 1 pg/µL. We combined the RAA-CRISPR/Cas12a with a lateral flow dipstick, which also showed specific, high sensitivity, and fast characters. In conclusion, the RAA-CRISPR/Cas12a assay has great potential to be a method for early diagnosis and on-site detection of C. parasitica, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of Cryphonectria parasitica was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor CpSge1. The detection system was performed at a constant temperature condition of 37°C, which provides important support for the diagnosis of chestnut blight diseases in the field.},
}

*Ascomycota/genetics/isolation & purification
*Plant Diseases/microbiology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Sensitivity and Specificity
Fagaceae/microbiology
Fungal Proteins/genetics
Recombinases/metabolism/genetics
China
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41075360,
year = {2026},
author = {Rahimi, M and Schreiber, M and Habermann, H and Haake, L and Miskel, D and Tesfaye, D and Held-Hoelker, E and Brenig, B and Benedetti, C and Hoelker, M},
title = {Efficient genome editing of bovine in vitro derived zygotes via Cas9 RNP-electroporation using extended-stored bovine ovaries.},
journal = {Theriogenology},
volume = {250},
number = {},
pages = {117703},
doi = {10.1016/j.theriogenology.2025.117703},
pmid = {41075360},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; Female ; *Gene Editing/veterinary/methods ; *Electroporation/veterinary/methods ; *Zygote/physiology ; *Ovary/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {Bovine embryos serve as a relevant model for human preimplantation development and are increasingly used in genome editing research. Although CRISPR-Cas9 electroporation in zygotes offers a promising approach, challenges with respect to editing efficiency and mosaicism remain a challenge. However, traditional in vitro fertilization (IVF) workflows enforce highly inconvenient electroporation times of zygotes usually around midnight. This study therefore aimed to investigate the feasibility of using bovine zygotes derived from extended-stored slaughterhouse derived ovaries (Phosphate buffered saline, 14-18 °C, 20-22 h) with particular attention to the strength of the voltage field during electroporation and the Cas9-RNP concentration with respect to embryonic development and genome editing efficiency. Oocytes obtained under these conditions demonstrated in vitro developmental competence similar to those derived from fresh ovaries, with comparable cleavage and blastocyst formation rates. Electroporation conducted 10 h post-insemination at voltages of 15V, 20V, and 25V revealed that 25V yielded the highest blastocyst editing rate (40.7 %) while maintaining acceptable developmental rates. Increasing voltage to 30V and 35V further enhanced blastocyst editing rate (66.7 % and 67.9 %, respectively), with a greater proportion of blastocyst showing a full edit. While higher voltages reduced developmental rates, the optimal voltage for electroporation was found to be 30V for bovine zygotes derived from extended stored ovaries. Using that setting, contrasting Cas9-RNP concentrations (6 μM vs. 3 μM) were tested. A concentration of 6 μM resulted in higher editing rates compared to 3 μM (81.5 % vs. 60.0 %), although the distribution of monoallelic, biallelic, and mosaic modifications did not differ significantly. Taken together, this study confirmed for the first time that zygotes from extend-stored bovine ovaries are suitable for genome editing via CRISPR Cas9-RNP electroporation. Mosaicism, however, remained prevalent, underscoring the need for strategies to improve the precision and consistency of embryo editing.},
}

Animals
Cattle/embryology
Female
*Gene Editing/veterinary/methods
*Electroporation/veterinary/methods
*Zygote/physiology
*Ovary/physiology
*CRISPR-Cas Systems
Fertilization in Vitro/veterinary
CRISPR-Associated Protein 9
Ribonucleoproteins/genetics

@article {pmid41036840,
year = {2025},
author = {Diaz, B and Krishna, R and Schoeniger, JS and Mageeney, CM},
title = {Exploring phage-host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193625},
pmid = {41036840},
issn = {2165-0497},
support = {LDRD//Sandia National Laboratories/ ; InCoGenTEC//U.S. Department of Energy/ ; },
mesh = {*Bacteriophages/physiology/genetics ; *Burkholderia cepacia complex/virology/genetics ; Genomics/methods ; Transcriptome ; Gene Expression Profiling ; CRISPR-Cas Systems ; Host Specificity ; Humans ; Host-Pathogen Interactions/genetics ; },
abstract = {Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.IMPORTANCEBurkholderia cepacia complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. Burkholderia bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic Burkholderia bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in Burkholderia cenocepacia K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing Burkholderia bacteria.},
}

*Bacteriophages/physiology/genetics
*Burkholderia cepacia complex/virology/genetics
Genomics/methods
Transcriptome
Gene Expression Profiling
CRISPR-Cas Systems
Host Specificity
Humans
Host-Pathogen Interactions/genetics

@article {pmid40996288,
year = {2025},
author = {Boucher, MJ and Madhani, HD},
title = {CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193525},
pmid = {40996288},
issn = {2165-0497},
support = {R01AI100272//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Cryptococcus neoformans/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; *Plasmids/genetics ; *Selection, Genetic ; Drug Resistance, Fungal/genetics ; Nucleosides ; },
abstract = {Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.},
}

*Cryptococcus neoformans/genetics/drug effects
*CRISPR-Cas Systems
Humans
*Plasmids/genetics
*Selection, Genetic
Drug Resistance, Fungal/genetics
Nucleosides

@article {pmid40089804,
year = {2025},
author = {Zhu, J and Zhang, Y and Zhang, M and Hong, Y and Sun, C and Guo, Y and Yin, H and Lv, C and Guo, B and Wang, F and Xu, R},
title = {Natural variation and CRISPR/Cas9 gene editing demonstrate the role of a group VII ethylene response factor, HvERF62, in regulation of barley waterlogging tolerance.},
journal = {Journal of experimental botany},
volume = {76},
number = {17},
pages = {5071-5085},
doi = {10.1093/jxb/eraf101},
pmid = {40089804},
issn = {1460-2431},
support = {32301871//National Natural Science Foundation of China/ ; 2022YFD2301302//National key research and development plant/ ; },
mesh = {*Hordeum/genetics/physiology/metabolism ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Ethylenes/metabolism ; Genome-Wide Association Study ; Quantitative Trait Loci ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Waterlogging stress is a serious problem for plants throughout the world, inhibiting plant growth and development and even leading in severe circumstances to plant death. Ethylene-responsive VII (ERFVII) is a key transcription factor regulating plant waterlogging tolerance. In this study, a major quantitative trait locus influencing chlorophyll content under waterlogging stress was detected on chromosome 4H through genome-wide association study using a natural population of barley (Hordeum vulgare). Using RNA-Seq analysis, the group VII ethylene response factor HvERF62 was identified as the candidate gene. CRISPR/Cas9-guided knockout HvERF62 mutants showed sensitivity to waterlogging, with lower chlorophyll content, fewer adventitious roots, and lower root activity. Physiological and transcriptomic analyses showed that HvERF62 plays an important role in aerenchyma formation, reactive oxygen species homeostasis, and carbohydrate accumulation under waterlogging stress, and regulates waterlogging tolerance through starch and sucrose metabolism, the mitogen-activated protein kinase signaling pathway, plant hormone signal transduction, and glycolysis/gluconeogenesis signaling. Haplotype analysis showed that the amino acid sequence of the waterlogging-intolerant haplotype Hap3 was terminated prematurely. This study provides a new genetic resource and a relevant marker for identification of waterlogging-sensitive material and deepens our understanding of the molecular mechanisms of waterlogging response in barley.},
}

*Hordeum/genetics/physiology/metabolism
*Plant Proteins/genetics/metabolism
*CRISPR-Cas Systems
Gene Editing
Ethylenes/metabolism
Genome-Wide Association Study
Quantitative Trait Loci
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant

@article {pmid41184108,
year = {2026},
author = {Abir, AH and Benz, J and Frey, B and Bruns, H and Krönke, G and Gaipl, US and Schober, K and Mougiakakos, D and Mielenz, D},
title = {Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation.},
journal = {Life science alliance},
volume = {9},
number = {1},
pages = {},
doi = {10.26508/lsa.202503315},
pmid = {41184108},
issn = {2575-1077},
mesh = {Humans ; *Glycolysis ; Glycosylation ; *STAT1 Transcription Factor/metabolism/genetics ; *Th1 Cells/metabolism/immunology ; Interferon-gamma/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Editing ; },
abstract = {T helper (Th) cell lineages are linked to metabolism, but precise mechanisms in human Th1 cells remain unclear. We addressed this question by in vitro stimulation and CRISPR/Cas9-mediated gene editing. Metabolic profiling revealed enhanced glycolytic activity in Th1 versus non-polarized cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, lactate secretion, NADH abundance, and elevated glycolysis-dependent anabolic activity. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 or SHP2 activity and STAT1 abundance, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. O-glycosylation of STAT1 via O-glycosyltransferase was pivotal in modulating STAT1 activity. Pharmaceutical O-glycosyltransfer-ase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells.},
}

Humans
*Glycolysis
Glycosylation
*STAT1 Transcription Factor/metabolism/genetics
*Th1 Cells/metabolism/immunology
Interferon-gamma/metabolism
Phosphorylation
CRISPR-Cas Systems
Cell Differentiation
Gene Editing

@article {pmid41183413,
year = {2025},
author = {Chen, H and Zeng, Z and Wei, Y and Huang, H and Negahdary, M and Han, X and Lin, Y and He, L and Song, F and Wan, Y},
title = {Dynamic biosensing enables amplifier-collateral-cleavage enhancement for pathogen diagnostic.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118158},
doi = {10.1016/j.bios.2025.118158},
pmid = {41183413},
issn = {1873-4235},
abstract = {While CRISPR-Cas system and dynamic DNA nanotechnology have been extensively applied to mainstream biomedical domains, including gene editing, biochemical analysis, and molecular imaging-current approaches remain constrained by limitations in addressing increasingly nuanced and specialized experimental scenarios. Here, we report that CRISPR-CasΦ possesses a unique collateral-cleavage blockade characteristic: CasΦ is unable to recognize the "TTN" sequence in the loop region at the 3' end of stem-loop DNA, resulting in the blockade of collateral-cleavage activity. Leveraging this discovery, we successfully designed and customized two back-end signal amplifiers for biosensing by integrating dynamic DNA sensing studies of CasΦ. Based on these two specialized stem-loop amplifiers, we further developed the Amplifier-collateral-cleavage enhancement (ACE) method, achieving exponential signal amplification. Clinical validation using 112 urine samples demonstrated that ACE exhibits 98.8 % sensitivity and 90 % specificity. These findings highlight the potential of CasΦ dynamic sensing and establish a bridge for future integration of dynamic DNA technology and CRISPR systems.},
}

@article {pmid41124025,
year = {2025},
author = {Su, F and Zhang, H and Ren, C and Jiang, Y and Qiao, Y and Zhang, S and Liu, J and Li, S and Li, Y},
title = {MnO2@Mn3O4 Heterojunction as a Coreactant Catalyst Collaborated with T-Shaped DNA Cycling-CRISPR/Cas12a Cascade Amplification for Locus-Specific N6-Methyladenosine RNA Modification Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {43},
pages = {24165-24175},
doi = {10.1021/acs.analchem.5c04791},
pmid = {41124025},
issn = {1520-6882},
mesh = {*Adenosine/analogs & derivatives/analysis ; *Manganese Compounds/chemistry ; *Oxides/chemistry ; *Biosensing Techniques/methods ; Humans ; *RNA/analysis/chemistry ; Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Catalysis ; Gold/chemistry ; Nucleic Acid Amplification Techniques ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; Luminescent Measurements ; HeLa Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {As the predominant RNA modification, N6-methyladenosine (m[6]A) is recognized to play pivotal regulatory roles in fundamental cellular functions and oncogenic processes. However, the precise analysis of site-specific m[6]A modifications continues to present significant challenges. In this work, a unique electrochemiluminescence (ECL) biosensor for the locus-specific detection of m[6]A in RNA was developed in the first utilization of a MnO2@Mn3O4 heterojunction as a coreaction catalyst in collaboration with a T-shaped DNA cycling-CRISPR/Cas12a cascade amplification strategy. The MnO2@Mn3O4 heterojunction was observed to significantly enhance coreactant catalytic activity, yielding a 7.3-fold increase in the ECL intensity of the gold nanoparticles (AuNPs)/MnO2@Mn3O4/(2,2'-bipyridine) dichlororuthenium(II) (Ru(bpy)3[2+])/Nafion/GCE compared to the AuNPs/Ru(bpy)3[2+]/Nafion/GCE. Sequentially, the T-shaped DNA cycling amplification strategy effectively converted the target m[6]A RNA into an amplified biosignal, further enhanced by a CRISPR/Cas12a signal amplification system mediated by framework nucleic acid (FNA)-based photocontrollable nucleic acid protection, ensuring the sensitivity and specificity of m[6]A RNA. The integration of the triple signal amplification strategy achieved detection limits as low as 0.05 pM (S/N = 3) for a linearity spanning from 100 fM to 100 nM. The proposed ECL biosensor has been applied in detecting site-specific m[6]A modifications in total real RNA samples extracted from HeLa cells, demonstrating its promising applications for clinical diagnosis.},
}

*Adenosine/analogs & derivatives/analysis
*Manganese Compounds/chemistry
*Oxides/chemistry
*Biosensing Techniques/methods
Humans
*RNA/analysis/chemistry
Electrochemical Techniques/methods
*CRISPR-Cas Systems
*DNA/chemistry/genetics
Metal Nanoparticles/chemistry
Catalysis
Gold/chemistry
Nucleic Acid Amplification Techniques
CRISPR-Associated Proteins/genetics
Limit of Detection
Luminescent Measurements
HeLa Cells
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41084820,
year = {2025},
author = {Ma, AX and Zhang, Q and Zhang, MY and Liu, SL and Wang, ZG and Pang, DW},
title = {One-step naked-eye fluorescence detection of viruses using quantum dot-magnetic beads coupled with CRISPR/Cas12a.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {89},
pages = {17432-17435},
doi = {10.1039/d5cc03645c},
pmid = {41084820},
issn = {1364-548X},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems ; Humans ; Fluorescence ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Simplexvirus/isolation & purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {This work constructs quantum dot-magnetic beads coupled with CRISPR/Cas12a for naked-eye detection of herpes simplex virus. The system integrates the high brightness of quantum dots and trans-cleavage activity of Cas12a, enabling rapid, simple, and accurate viral detection.},
}

*Quantum Dots/chemistry
*CRISPR-Cas Systems
Humans
Fluorescence
*CRISPR-Associated Proteins/metabolism/chemistry
*Simplexvirus/isolation & purification
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40954319,
year = {2025},
author = {Wei, D and Cheng, P and Song, Z and Liu, Y and Xu, X and Huang, X and Wang, X and Zhang, Y and Shu, W and Wei, Y},
title = {AI-guided Cas9 engineering provides an effective strategy to enhance base editing.},
journal = {Molecular systems biology},
volume = {21},
number = {11},
pages = {1563-1580},
pmid = {40954319},
issn = {1744-4292},
support = {12331018//MOST | National Natural Science Foundation of China (NSFC)/ ; 32401220//MOST | National Natural Science Foundation of China (NSFC)/ ; PT2025-03-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; RC2025-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; CXPJJH122006-1014//CHEN XIAO-PING FOUNDATION FOR THE DEVELOPMENT OF SCIENCE AND TECHNOLOGY OF HUBEI PROVINCE/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Protein Engineering/methods ; *Artificial Intelligence ; Point Mutation ; Cell Line, Tumor ; HEK293 Cells ; },
abstract = {Precise genome editing is crucial for functional studies and therapies. Base editors, while powerful, require optimization for efficiency. Meanwhile, emerging protein design methods and protein language models have driven efficient and intelligent protein engineering. In this study, we employed the Protein Mutational Effect Predictor (ProMEP) to predict the effects of single-site saturated mutations in Cas9 protein, using AncBE4max as the prototype to construct and test 18 candidate point mutations. Based on this, we further predicted combinations of multiple mutations and successfully developed a high-performance variant AncBE4max-AI-8.3, achieving a 2-3-fold increase in average editing efficiency. Introducing the engineered Cas9 into CGBE, YEE-BE4max, ABE-max, and ABE-8e improved their editing performance. The same strategy also substantially improves the efficiencies of HF-BEs. Stable enhancement in editing efficiency was also observed across seven cancer cell lines and human embryonic stem cells. In conclusion, we validated that AI models can serve as more effective protein engineering tools, providing a universal improvement strategy for a series of gene editing tools.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*Protein Engineering/methods
*Artificial Intelligence
Point Mutation
Cell Line, Tumor
HEK293 Cells

@article {pmid40878190,
year = {2025},
author = {Tian, L and Gao, Y and Lu, Y and Xu, F and Feng, Z and Zi, L and Deng, Z and Yang, J},
title = {Modular RCA-CRISPR/Cas12a amplification on a multi-volume SlipChip for ultrafast, single-copy quantification of circRNA and miRNA in ovarian cancer.},
journal = {Lab on a chip},
volume = {25},
number = {22},
pages = {5762-5776},
doi = {10.1039/d5lc00585j},
pmid = {40878190},
issn = {1473-0189},
mesh = {Humans ; *MicroRNAs/genetics/analysis ; *RNA, Circular/genetics/analysis ; Female ; *Ovarian Neoplasms/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Lab-On-A-Chip Devices ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The aberrant expression of RNAs in ovarian cancer (OC) progression highlights their potential as clinical biomarkers. However, rapid and accurate quantification of these RNAs in biosamples remains a significant challenge. In this study, we develop a modular isothermal rolling circle amplification (RCA)-activated Cas12a loop-enhanced (MIRACLE) amplification method for circRNA and miRNA quantification without the need of reverse transcription. In this design, isothermal amplification of modular DNA can be initiated by target-specific RCA primers or miRNAs, with the amplification products subsequently recognized by the Cas12a system to generate measurable signals. When integrated with a multi-volume sliding chip (SlipChip) platform, this MIRACLE method enables portable, rapid and ultra-sensitive quantification of these two types of RNA. Under optimized conditions, this platform exhibits detection limits of 0.125 copies per μL for circRNA and 0.326 copies per μL for miRNA, covering a 5-log dynamic range from 10[-1] to 10[3] copies per μL within 35 min. The platform was validated using OC cell lines and clinical blood samples. It successfully profiled OC RNA biomarkers (hsa_circ_0049101 and hsa-miR-338-3p) and effectively distinguished between early and advanced stages of OC. These results show a strong correlation with RT-qPCR (R[2] = 0.953 for circRNA and R[2] = 0.947 for miRNA). This work establishes a versatile CRISPR-microfluidic platform for cancer diagnosis. Its modular design allows for adaptation to detect other cancer-related RNA biomarkers, thereby addressing critical needs in precision oncology.},
}

Humans
*MicroRNAs/genetics/analysis
*RNA, Circular/genetics/analysis
Female
*Ovarian Neoplasms/genetics
*Nucleic Acid Amplification Techniques/instrumentation
*CRISPR-Cas Systems
Cell Line, Tumor
*Lab-On-A-Chip Devices
*CRISPR-Associated Proteins/metabolism/genetics
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41183015,
year = {2025},
author = {Keuthen, H and Pozhydaieva, N and Höfer, K},
title = {Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69022},
pmid = {41183015},
issn = {1940-087X},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; *Mixed Function Oxygenases/genetics/metabolism ; Mutagenesis ; DNA, Viral/genetics/chemistry ; *Mutagenesis, Site-Directed/methods ; },
abstract = {Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions. However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage. In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*Bacteriophages/genetics
*Mixed Function Oxygenases/genetics/metabolism
Mutagenesis
DNA, Viral/genetics/chemistry
*Mutagenesis, Site-Directed/methods

@article {pmid41182907,
year = {2025},
author = {Pantoja-Alonso, MA and Camas-Reyes, JA and Cano-Segura, R and Cárdenas-Aquino, MDR and Martínez-Antonio, A},
title = {A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {11},
pages = {},
doi = {10.1099/mic.0.001628},
pmid = {41182907},
issn = {1465-2080},
mesh = {*Bacteria/genetics/metabolism ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; *Genome, Bacterial ; Genomics/methods ; Gene Editing ; Metabolic Engineering/methods ; Synthetic Biology/methods ; },
abstract = {Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.},
}

*Bacteria/genetics/metabolism
*Genetic Engineering/methods
CRISPR-Cas Systems
*Genome, Bacterial
Genomics/methods
Gene Editing
Metabolic Engineering/methods
Synthetic Biology/methods

@article {pmid41182905,
year = {2025},
author = {Lamb, CH and Riesle-Sbarbaro, S and Prescott, JB and Te Velthuis, AJW and Myhrvold, C and Nilsson-Payant, BE},
title = {Amplification-free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
doi = {10.1099/jgv.0.002169},
pmid = {41182905},
issn = {1465-2099},
mesh = {Animals ; *RNA, Viral/genetics ; Humans ; *Influenza A virus/genetics/isolation & purification ; Sensitivity and Specificity ; *Orthohantavirus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Zoonoses/virology/diagnosis ; },
abstract = {Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.},
}

Animals
*RNA, Viral/genetics
Humans
*Influenza A virus/genetics/isolation & purification
Sensitivity and Specificity
*Orthohantavirus/genetics/isolation & purification
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Zoonoses/virology/diagnosis

@article {pmid41182611,
year = {2026},
author = {Chiurillo, MA and Ahmed, M and González, C and Rosón, JN and Das, A and Lander, N},
title = {Cloning-Free Genome Editing by CRISPR/T7RNAP/Cas9 in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2982},
number = {},
pages = {59-76},
pmid = {41182611},
issn = {1940-6029},
mesh = {*Trypanosoma cruzi/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA-Directed RNA Polymerases/genetics ; *Viral Proteins/genetics ; Cloning, Molecular ; Humans ; *Genome, Protozoan ; Gene Knockout Techniques ; },
abstract = {The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.},
}

*Trypanosoma cruzi/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*DNA-Directed RNA Polymerases/genetics
*Viral Proteins/genetics
Cloning, Molecular
Humans
*Genome, Protozoan
Gene Knockout Techniques

@article {pmid41182029,
year = {2025},
author = {Ashworth, KE and Zhang, J and D'Amata, C and Héon, E and Ballios, BG},
title = {USH2A-Mutated Human Retinal Organoids Model Rod-Cone Dystrophy.},
journal = {Investigative ophthalmology & visual science},
volume = {66},
number = {14},
pages = {2},
doi = {10.1167/iovs.66.14.2},
pmid = {41182029},
issn = {1552-5783},
mesh = {Humans ; *Organoids/pathology/metabolism ; Induced Pluripotent Stem Cells/pathology/metabolism ; *Mutation ; *Extracellular Matrix Proteins/genetics/metabolism ; *Cone-Rod Dystrophies/genetics/pathology/metabolism ; Retinal Rod Photoreceptor Cells/pathology ; *Retinitis Pigmentosa/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP), a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans, and its earliest pathogenic events remain unknown. Here, we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A-/- induced pluripotent stem cells.

METHODS: We assessed organoids for cellular, molecular, and morphological defects using serial live imaging and whole organoid and fixed section analyses.

RESULTS: Both patient-derived and isogenic-USH2A-/- organoids showed preferential rod photoreceptor loss followed by widespread degeneration, consistent with the clinical phenotype. Additionally, isogenic-USH2A-/- organoids showed early defects in proliferation and structure.

CONCLUSIONS: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP, and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features, we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies.},
}

Humans
*Organoids/pathology/metabolism
Induced Pluripotent Stem Cells/pathology/metabolism
*Mutation
*Extracellular Matrix Proteins/genetics/metabolism
*Cone-Rod Dystrophies/genetics/pathology/metabolism
Retinal Rod Photoreceptor Cells/pathology
*Retinitis Pigmentosa/genetics/pathology
CRISPR-Cas Systems

@article {pmid41178730,
year = {2025},
author = {Avaro, AS and Santiago, JG},
title = {Engineering guidelines for CRISPR diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04206b},
pmid = {41178730},
issn = {1364-548X},
abstract = {This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.},
}

@article {pmid41178318,
year = {2025},
author = {Leung, CY and Wimmer, EA and Ahmed, HMM},
title = {Synthetic biology approaches to generate temperature-sensitive alleles for the Sterile Insect Technique.},
journal = {Insect science},
volume = {},
number = {},
pages = {},
doi = {10.1111/1744-7917.70186},
pmid = {41178318},
issn = {1744-7917},
support = {ResearchcooperationLowerSaxony-Israel//Niedersächsisches Ministerium für Wissenschaft und Kultur/ ; Project D44003//International Atomic Energy Agency/ ; },
abstract = {The Sterile Insect Technique (SIT) is an environmentally friendly, sustainable pest control approach, which uses large-scale releases of sterile insects to suppress or eradicate target populations through infertile matings. The efficiency of SIT is enhanced by male-only releases requiring genetic sexing strains (GSSs) that are classically based on selectable recessive visible markers or temperature-sensitive lethal (tsl) mutations and a rescue by a wild-type allele translocated to the male-determining chromosome. The transfer of identified or designed temperature-sensitive alleles might allow the generation of neoclassical GSSs in additional SIT target species. By using precise genome-editing tools, such as CRISPR/Cas, the creation of specific mutations in target genes and the integration of a wild-type copy is feasible without the introduction of foreign DNA. This might ease regulation of neoclassical GSSs, since they are not considered transgenic. However, integration and expression of genes at male-determining loci or chromosomes is not reliably established. Therefore, additional strategies to link temperature-sensitive phenotypes to female development are required, which could be achieved by targeting genes involved in dosage compensation or sex determination. To create temperature-sensitive alleles, rational protein design using advanced modeling and prediction tools to evaluate and tailor the effect of mutations on protein stability and temperature sensitivity can be used. In addition, emerging synthetic biology strategies such as temperature-inducible N-degrons or temperature-sensitive inteins provide powerful tools to generate temperature sensitivity. Such approaches should enable conditional control over proteins causing female lethality or sex conversion and therefore promise straightforward generic approaches to generate GSSs for male-only production in SIT target species.},
}

@article {pmid41177343,
year = {2025},
author = {Rahimian, M and Panahi, B},
title = {Genomic insights into Erwinia amylovora prophages: Diversity, defense strategies, and phage-host coevolution.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105846},
doi = {10.1016/j.meegid.2025.105846},
pmid = {41177343},
issn = {1567-7257},
abstract = {Erwinia amylovora, the causative agent of fire blight in Rosaceae plants like apple and pear, is a major agricultural pathogen responsible for significant economic losses. This gram-negative phytopathogen has the potential to acquire antibiotic resistance and virulence genes, a situation that becomes more severe and restricts treatment options. Using an in silico approach, we analyzed 268 E. amylovora genomes and identified seven high-quality temperate prophages, all classified under Caudoviricetes, with average genome sizes of 44.2 kbp and 51 % GC content. These prophages exhibited unique genomic features, including tRNA genes (Ph-Ea644), anti-defense systems like ardc (Ph-Ea6-96), and regulatory/lysis genes (Ph-EaFC01). Comparative genomics and phylogenetic analyses grouped them into five clades, with Ph-Ea4-96, Ph-Ea3-97, and Ph-Ea2-97 being genetically identical. Functional annotation revealed streptomycin resistance genes and a CAZyme (GH23) in Ph-Ea7-3, virulence factors (e.g., alginate biosynthesis proteins), and six auxiliary metabolic genes (AMGs) linked to metabolic adaptation. Additionally, Ph-Ea644 encoded a cell wall-binding receptor protein. The prophages also carried defense systems (Gabija, CBASS) and 31 anti-CRISPR proteins (ACRs), suggesting evasion of host immunity. CRISPR-Cas analysis indicated fewer arrays and spacers in prophage-containing strains, underscoring CRISPR's role in lysogeny resistance. These findings highlight the genomic plasticity of E. amylovora prophages, their interactions with bacterial defenses, and their potential influence on pathogen evolution. This study enhances understanding of temperate phages in agricultural pathogens and underscores challenges in phage-based biocontrol strategies.},
}

@article {pmid41177179,
year = {2025},
author = {Bacci, L and Pollutri, D and Ripa, IJ and D'Andrea, M and Marchand, V and Motorin, Y and Hesse, AM and Couté, Y and Filipek, K and Penzo, M},
title = {Ribosomal protein L5 (RPL5/uL18) I60V mutation is associated to increased translation and modulates drug sensitivity in T-cell acute lymphoblastic leukemia cells.},
journal = {Biochemical pharmacology},
volume = {},
number = {},
pages = {117497},
doi = {10.1016/j.bcp.2025.117497},
pmid = {41177179},
issn = {1873-2968},
abstract = {Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10 % of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels-including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B-as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies.},
}

@article {pmid41176953,
year = {2025},
author = {Lankireddy, SV and Lekkala, S and Khadgi, A and Sripathi, VR and Janga, MR},
title = {Molecular biology of Cotton Leafroll Dwarf Virus (CLRDV) and potential application of CRISPR-Cas technology for developing virus-resistant cotton.},
journal = {Virology},
volume = {614},
number = {},
pages = {110730},
doi = {10.1016/j.virol.2025.110730},
pmid = {41176953},
issn = {1096-0341},
abstract = {Cotton leafroll dwarf virus (CLRDV) poses an increasing threat to global cotton production. Transmitted by the cotton aphid (Aphis gossypii) in a persistent, circulative manner, CLRDV exhibits a wide geographical distribution, with documented presence in South America, Africa, Asia, and the USA. Infection can result in either cotton blue disease (CBD) in South America or cotton leafroll dwarf disease (CLRDD) in the USA, both of which are associated with CLRDV. The considerable genetic diversity and frequent recombination events within CLRDV populations contribute to this symptom variability and complicate both diagnosis and management. While resistant cultivars have reduced disease impact in South America, these lines remain susceptible to emerging US strains, underscoring the urgent need for region-specific resistance breeding. Current molecular diagnostics rely on RT-PCR, but there is a need for rapid, field-deployable detection tools. Recent advances, such as CRISPR-Cas13a based SHERLOCK assays, offer sensitive and specific detection of CLRDV, with potential for on-site applications. Efficient screening techniques, supported by next-generation sequencing and transcriptomics, are essential for identifying novel resistance sources and elucidating virus-host interactions. CRISPR-based genome editing holds significant promise, as demonstrated in other crops. Targeted disruption of host susceptibility genes using CRISPR-Cas9, or direct degradation of viral genomes with RNA-targeting systems such as Cas12/Cas13, could offer durable, broad-spectrum resistance. By integrating molecular virology, high-throughput genomics, and precision gene editing, this review outlines a roadmap for translating these advances into sustainable, field-level solutions for CLRDV management and long-term cotton productivity.},
}

@article {pmid41176802,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Erratum to: Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {10},
pages = {1450},
doi = {10.1134/S0006297925100013},
pmid = {41176802},
issn = {1608-3040},
}

@article {pmid41175344,
year = {2025},
author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Denise Fischer, K and Almolla, O and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Boccalatte, F and Wang, E and Imig, J},
title = {RNA binding of GAPDH controls transcript stability and protein translation in acute myeloid leukemia.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-23},
doi = {10.1080/15476286.2025.2580180},
pmid = {41175344},
issn = {1555-8584},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism/genetics ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/metabolism/genetics ; 5' Untranslated Regions ; Cell Line, Tumor ; *RNA Stability ; Protein Binding ; Gene Expression Regulation, Leukemic ; Cell Proliferation ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; },
abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukaemia (AML) RBPs are key regulators of tumour proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/metabolism/pathology
*Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism/genetics
*Protein Biosynthesis
*RNA, Messenger/genetics/metabolism
RNA-Binding Proteins/metabolism/genetics
5' Untranslated Regions
Cell Line, Tumor
*RNA Stability
Protein Binding
Gene Expression Regulation, Leukemic
Cell Proliferation
Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)

@article {pmid40958477,
year = {2025},
author = {Tao, XL and Lei, YM and Zhou, XM and Chen, ZP and Ma, Y and Ma, PY and Song, DQ and Zhuo, Y},
title = {Allosteric Activation of Cas12a via PAM Topological Engineering for Direct and Rapid Detection of Nucleases.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {45},
pages = {e202515521},
doi = {10.1002/anie.202515521},
pmid = {40958477},
issn = {1521-3773},
support = {22374123//National Natural Science Foundation of China/ ; SWU-XJLJ202303//The Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; Allosteric Regulation ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins ; },
abstract = {A contemporary question in the intensely active field of CRISPR-Cas12a-based molecular diagnostics is how to simplify the multistep conversion process for detecting nonnucleic acid targets. Herein we describe an allosteric Y-shaped DNA structure for Cas12a activation via protospacer-adjacent motif (PAM) topological engineering (Y-COPE) to achieve straightforward and diverse nuclease monitoring. The newly designed topological structure of the Y-COPE is characterized by a split PAM embedded at the three-way junction and protospacers flanking both sides. This unique spatial configuration of the PAM effectively prevents Cas12a activation. Upon target cleavage, the released truncated fragments can dynamically correct the PAM, which promptly restores the dsDNA conformation for Cas12a activation and accomplishes signal output. Theoretical calculation results revealed that, compared with the canonical dsDNA activator, in the Y-COPE, there was a 1.8 Å increase in the center distance between Lys595 of Cas12a and the PAM, which led to a 24.2 kcal mol[-1] increase in binding free energy. This clearly revealed the underlying inhibition mechanism of the topological configuration of the PAM for Cas12a activation. This study advances the understanding of the dynamic response of Cas12a to topological PAM conformations and introduces the universal concept of CRISPR-based nonnucleic acid detection to benefit the next-generation molecular diagnostics.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
Allosteric Regulation
CRISPR-Cas Systems
*DNA/chemistry/metabolism
*Endodeoxyribonucleases/metabolism/chemistry
Bacterial Proteins

@article {pmid40888991,
year = {2025},
author = {Lin, HK and Dai, J and Pusztai, L},
title = {Integrating large-scale in vitro functional genomic screen and multi-omics data to identify novel breast cancer targets.},
journal = {Breast cancer research and treatment},
volume = {214},
number = {3},
pages = {319-327},
pmid = {40888991},
issn = {1573-7217},
support = {BCRF-22-133, SAC220225//Breast Cancer Research Foundation Investigator Award (BCRF-22-133), Susan Komen Leadership Grant (SAC220225)/ ; },
mesh = {Humans ; Female ; *Breast Neoplasms/genetics/drug therapy/pathology ; *Genomics/methods ; DNA Copy Number Variations ; Cell Line, Tumor ; *Biomarkers, Tumor/genetics ; Mutation ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; CRISPR-Cas Systems ; Transcriptome ; Molecular Targeted Therapy ; Multiomics ; },
abstract = {PURPOSE: Our goal is to leverage publicly available whole transcriptome and genome-wide CRISPR-Cas9 screen data to identify and prioritize novel breast cancer therapeutic targets.

METHODS: We used DepMap dependency scores > 0.5 to identify genes that are potential therapeutic targets in 48 breast cancer cell lines. We removed genes that were pan-essential or were not expressed in TCGA breast cancer cohort. Genes were prioritized based on druggability using the Drug-Gene Interaction Database. Targets were defined separately for ER+, HER2+, and TNBC. A broader list of genes with dependency score > 0.25 were used to assess the associations between dependency scores and mutations and copy number variations (CNV) to identify potential synthetic lethal relationships and to map survival critical genes into biological pathways.

RESULTS: 66, 53, and 29 genes were prioritized as targets in ER+, HER2+, and TNBC, respectively. These included known actionable targets and many novel targets. ER+ included FOXA1, GATA3, LDB1, TRPS1, NAMPT, WDR26, and ZNF217; HER2+ cancers included STX4, HECTD1, and TBL1XR1; and TNBC included GFPT1 and GPX4. Synthetic lethal associations revealed 5 and 19 significant associations between potential survival critical genes and mutations in HER2+ and TNBC, respectively. For example, PIK3CA mutation increased dependency on NDUFS3 in HER2+ cancers, and CNTRL mutation increased dependency on electron transport chain (ETC) genes in TNBC. 329, 747, and 622 CNVs showed synthetic lethal association in ER+, HER2+, and TNBC, respectively.

CONCLUSION: We provide a genome-wide drug target prioritization list for breast cancer derived from integrated large-scale omics data.},
}

Humans
Female
*Breast Neoplasms/genetics/drug therapy/pathology
*Genomics/methods
DNA Copy Number Variations
Cell Line, Tumor
*Biomarkers, Tumor/genetics
Mutation
Gene Expression Regulation, Neoplastic
Gene Expression Profiling
CRISPR-Cas Systems
Transcriptome
Molecular Targeted Therapy
Multiomics

@article {pmid40590508,
year = {2025},
author = {Mohammadzadeh, R and Shahbazi, S and Khodaei, N and Sabzi, S},
title = {Emerging Therapeutic Strategies to Combat Antimicrobial Resistance in the Post-Antibiotic Era.},
journal = {Journal of basic microbiology},
volume = {65},
number = {11},
pages = {e70070},
doi = {10.1002/jobm.70070},
pmid = {40590508},
issn = {1521-4028},
mesh = {Humans ; *Bacterial Infections/therapy/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; *Drug Resistance, Bacterial/drug effects ; *Bacteria/drug effects/pathogenicity ; Probiotics/therapeutic use ; Bacteriophages ; Antimicrobial Peptides/therapeutic use/pharmacology ; Bacteriocins/therapeutic use ; Animals ; CRISPR-Cas Systems ; },
abstract = {Antimicrobial resistance (AMR) is a serious global health issue. This review aims to explore alternative therapeutic strategies for combating AMR. The goal is to evaluate emerging treatments that target resistant pathogens through novel mechanisms, bypassing the limitations of traditional antibiotics. Recent researches highlight several promising alternatives, including antibodies, antimicrobial peptides, bacteriocins, bacteriophages, and probiotics (in the clinical trials) and synthetic antimicrobial peptides, anti-virulence strategies, genetically modified phages, antibacterial oligonucleotides, CRISPR-Cas9, and predatory bacteria (in the research stage). These therapies demonstrate potential to overcome AMR by targeting specific bacterial mechanisms, reducing toxicity, and evading resistance. Alternative therapies for AMR present significant promise, offering new avenues for treatment. Despite challenges in optimization and delivery, these therapies could revolutionize the way bacterial infections are treated. Continued research is crucial to address hurdles and ensure these therapies can be safely and effectively implemented in clinical settings, shaping the future of infection management.},
}

Humans
*Bacterial Infections/therapy/microbiology/drug therapy
*Anti-Bacterial Agents/pharmacology/therapeutic use
*Drug Resistance, Bacterial/drug effects
*Bacteria/drug effects/pathogenicity
Probiotics/therapeutic use
Bacteriophages
Antimicrobial Peptides/therapeutic use/pharmacology
Bacteriocins/therapeutic use
Animals
CRISPR-Cas Systems

@article {pmid41174869,
year = {2025},
author = {Li, S and Zhang, B and Ma, P and Zhang, Y and Hu, Z and Wu, X and Chen, Q and Zhao, Y},
title = {The 13-lipoxygenase GmLOX6 is involved in JA biosynthesis and serves as a positive regulator of salt stress tolerance in soybean.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70550},
pmid = {41174869},
issn = {1365-313X},
support = {2023ZD04036//Biological Breeding-National Science and Technology Major Project/ ; 2021YFD1201104-02-02//National Key Research and Development Program of China/ ; 2023YFD2300101//National Key Research and Development Program of China/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U23A20192//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/physiology/enzymology/metabolism ; *Lipoxygenase/metabolism/genetics ; *Oxylipins/metabolism ; *Cyclopentanes/metabolism ; *Salt Tolerance/genetics/physiology ; *Plant Proteins/metabolism/genetics ; Salt Stress ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; alpha-Linolenic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Salinity represents a major abiotic stressor that significantly impairs soybean growth and yield. Although jasmonic acid (JA) has been firmly established as a key regulator of plant defense against salt stress, the precise functions of lipoxygenase (LOX) genes responsible for initiating JA biosynthesis remain poorly defined. Here, a comprehensive genome-wide analysis of the soybean LOX gene family was performed, and a detailed functional characterization of GmLOX6 was carried out. Subcellular localization confirmed that GmLOX6 is targeted to chloroplasts, while enzymatic assays demonstrated that it acts as a 13-LOX enzyme with a strong preference for α-linolenic acid as substrate. To clarify its role under salt stress, we generated both overexpression and CRISPR/Cas9-mediated knockout lines of soybean. Phenotypic and molecular evaluations revealed that GmLOX6 facilitates JA production under salt stress, thereby contributing to enhanced JA accumulation. This elevation in JA levels was associated with improved salt tolerance through multiple physiological adaptations, including the activation of antioxidant enzymes for the detoxification of reactive oxygen species (ROS), enhanced Na[+] extrusion to preserve ionic balance, and reinforced membrane stability. Moreover, GmRWP-RK11 was identified as a transcriptional repressor of GmLOX6. Functional disruption of GmRWP-RK11 via CRISPR/Cas9 conferred greater salt tolerance, further supporting its negative regulatory role. Collectively, these findings uncover a novel regulatory axis in which GmLOX6-mediated JA biosynthesis enhances soybean resistance to salinity through modulation of ROS homeostasis and Na[+] transport. These insights provide an expanded understanding of the transcriptional and biochemical mechanisms underpinning JA-driven stress adaptation in soybean.},
}

*Glycine max/genetics/physiology/enzymology/metabolism
*Lipoxygenase/metabolism/genetics
*Oxylipins/metabolism
*Cyclopentanes/metabolism
*Salt Tolerance/genetics/physiology
*Plant Proteins/metabolism/genetics
Salt Stress
Gene Expression Regulation, Plant
Reactive Oxygen Species/metabolism
Plant Growth Regulators/metabolism
Plants, Genetically Modified
alpha-Linolenic Acid/metabolism
CRISPR-Cas Systems

@article {pmid41174319,
year = {2026},
author = {Fatmi, MQ and Nadeem, A and Abbasov, M and Sajjad, M},
title = {Computational Methods to Engineer Cas Proteins for Efficient Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2979},
number = {},
pages = {279-300},
pmid = {41174319},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Molecular Dynamics Simulation ; *CRISPR-Cas Systems/genetics ; *Protein Engineering/methods ; *Computational Biology/methods ; Mutation ; *CRISPR-Associated Proteins/genetics/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genome editing through programmable, sequence-specific deoxyribonucleic acid (DNA) targeting. Yet, its broader application remains limited by off-target effects and context-dependent efficiency. To address these challenges, we present an integrated computational protocol with easy-to-do steps for researchers to guide the rational design of CRISPR/Cas variants with improved stability and specificity. The integrated workflow begins with coevolutionary coupling analysis to identify conserved and covarying residues critical for function. These residues are then evaluated for energetically favorable substitutions through mutant stability prediction, followed by network centrality analysis to evaluate the impact of mutations on intramolecular communication pathways, preserving key allosteric interactions. Finally, molecular dynamics (MD) simulations validate the structural integrity and dynamic behavior of the selected variants. Network analysis and molecular dynamics (MD) simulations are applied iteratively, allowing insights from MD to refine network-based evaluations and vice versa. This multiscale strategy offers a streamlined and systematic approach for engineering optimized Cas proteins for genome editing applications.},
}

*Gene Editing/methods
Molecular Dynamics Simulation
*CRISPR-Cas Systems/genetics
*Protein Engineering/methods
*Computational Biology/methods
Mutation
*CRISPR-Associated Proteins/genetics/chemistry

@article {pmid41173880,
year = {2025},
author = {Binenbaum, J and Adamkova, V and Fryer, H and Xu, L and Gorringe, N and Włodzimierz, P and Burns, R and Papikian, A and Jacobsen, SE and Henderson, IR and Harris, CJ},
title = {CRISPR targeting of H3K4me3 activates gene expression and unlocks centromere-proximal crossover recombination in Arabidopsis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9587},
pmid = {41173880},
issn = {2041-1723},
support = {ERC Starting Grant (TransPlantMemory)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/Z001749/1//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ERC Advanced Grant (EvoPanCen)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/X025306/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; URF\R1\201016//Royal Society/ ; doctoral studentship//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V003984/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Histones/metabolism/genetics ; *Gene Expression Regulation, Plant ; *Centromere/genetics/metabolism ; *Crossing Over, Genetic ; *CRISPR-Cas Systems/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Disease Resistance/genetics ; Meiosis/genetics ; Plants, Genetically Modified ; },
abstract = {H3K4me3 is a fundamental and highly conserved chromatin mark across eukaryotes, playing a central role in many genome-related processes, including transcription, maintenance of cell identity, DNA damage repair, and meiotic recombination. However, identifying the causal function of H3K4me3 in these diverse pathways remains a challenge, and we lack the tools to manipulate it for agricultural benefit. Here we use the CRISPR-based SunTag system to direct H3K4me3 methyltransferases in the model plant, Arabidopsis thaliana. Targeting of SunTag-SDG2 activates the expression of the endogenous reporter gene, FWA. We show that SunTag-SDG2 can be employed to increase pathogen resistance by targeting the H3K4me3-dependent disease resistance gene, SNC1. Meiotic crossover recombination rates impose a limit on the speed with which new traits can be transferred to elite crop varieties. We demonstrate that targeting of SunTag-SDG2 to low recombining centromeric regions can significantly stimulate proximal crossover formation. Finally, we reveal that the effect is not specific to SDG2 and is likely dependent on the H3K4me3 mark itself, as the orthogonal mammalian-derived H3K4me3 methyltransferase, PRDM9, produces a similar effect on gene expression with reduced off-target potential. Overall, our study supports an instructive role for H3K4me3 in transcription and meiotic recombination and opens the door to precise modulation of important agricultural traits.},
}

*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/genetics/metabolism
*Histones/metabolism/genetics
*Gene Expression Regulation, Plant
*Centromere/genetics/metabolism
*Crossing Over, Genetic
*CRISPR-Cas Systems/genetics
Histone-Lysine N-Methyltransferase/genetics/metabolism
Disease Resistance/genetics
Meiosis/genetics
Plants, Genetically Modified

@article {pmid41173850,
year = {2025},
author = {Brown, BC and Tokolyi, A and Morris, JA and Lappalainen, T and Knowles, DA},
title = {Large-scale causal discovery using interventional data sheds light on gene network structure in k562 cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9628},
pmid = {41173850},
issn = {2041-1723},
support = {K99HG012373//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K99HG012792//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AG057422//U.S. Department of Health & Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; U01AG068880//U.S. Department of Health & Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; R01MH106842//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
mesh = {Humans ; *Gene Regulatory Networks ; K562 Cells ; CRISPR-Cas Systems ; },
abstract = {Inference of directed biological networks is an important but notoriously challenging problem. The recent proliferation of large-scale CRISPR perturbation data provides a new opportunity to tackle this problem by leveraging the transcriptional response to the presence of a gene-targeting guide. Here, we introduce inverse sparse regression (inspre), an approach to learning causal networks that leverages large-scale intervention-response data. Applied to 788 genes from the genome-wide perturb-seq dataset, inspre discovers a network with small-world and scale-free properties. We integrate our network estimate with external data, finding relationships between gene eigencentrality and both measures of gene essentiality and gene expression heritability. Our analysis helps to elucidate the structure of networks that may underlie complex traits.},
}

Humans
*Gene Regulatory Networks
K562 Cells
CRISPR-Cas Systems

@article {pmid39832721,
year = {2025},
author = {Mao, X and Xiong, J and Cai, M and Wang, C and He, Q and Wang, B and Chen, J and Xiao, Z and Wang, B and Han, S and Zhang, Y},
title = {SCARB1 links cholesterol metabolism-mediated ferroptosis inhibition to radioresistance in tumor cells.},
journal = {Journal of advanced research},
volume = {77},
number = {},
pages = {207-219},
doi = {10.1016/j.jare.2025.01.026},
pmid = {39832721},
issn = {2090-1224},
mesh = {*Ferroptosis/genetics ; Humans ; *Radiation Tolerance/genetics ; *Cholesterol/metabolism ; *Scavenger Receptors, Class B/metabolism/genetics ; Lipid Peroxidation ; Cell Line, Tumor ; Animals ; *Neoplasms/metabolism/radiotherapy/pathology/genetics ; Mice ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; },
abstract = {INTRODUCTION: Ferroptosis is an iron-dependent form of cell death triggered by the excessive accumulation of lipid peroxides. Understanding the regulatory mechanisms of ferroptosis and developing strategies to target this process hold significant clinical applications in tumor therapy.

OBJECTIVE: Our study aims to search for novel candidate genes involved in the regulation of ferroptosis and to investigate their mechanism of action in ferroptosis and tumor therapy.

METHODS: We employed a CRISPR-Cas9 library to perform a genome-wide screen under ferroptosis inducer treatment conditions, revealing Scavenger Receptor Class B Member 1(SCARB1) as a novel candidate gene involved in ferroptosis regulation. Subsequently, lipidomic analyses, metabolic interventions, and relevant cellular experimental analyses were performed to elucidate the role of SCARB1 in ferroptosis, lipid peroxidation, and tumor therapy.

RESULTS: Our study confirmed that SCARB1 significantly inhibits ferroptosis and lipid peroxidation induced by ferroptosis inducers. Mechanistically, SCARB1 inhibits ferroptosis through the regulation of cholesterol metabolism, and the upregulation of CoQ10 level is demonstrated to mediate the suppression of ferroptosis by SCARB1 after lipidomic analysis and metabolic intervention. Interestingly, SCARB1 exerts a tumor suppressive effect regarding tumor growth, migration and invasion, which is possibly independent of ferroptosis regulation. However, SCARB1 promotes radioresistance through the upregulation of cholesterol metabolism and inhibition of ferroptosis, while the combination of ferroptosis inducers can overcome radioresistance in tumor cells with high SCARB1 expression.

CONCLUSION: This study establishes a theoretical foundation for the regulation of ferroptosis by SCARB1 and highlights the potential of targeting lipid metabolism to overcome radioresistance in cancer therapy. The identification of SCARB1 as a key player in ferroptosis and its dual role in tumor suppression and radioresistance provides new avenues for therapeutic intervention in cancer treatment.},
}

*Ferroptosis/genetics
Humans
*Radiation Tolerance/genetics
*Cholesterol/metabolism
*Scavenger Receptors, Class B/metabolism/genetics
Lipid Peroxidation
Cell Line, Tumor
Animals
*Neoplasms/metabolism/radiotherapy/pathology/genetics
Mice
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic

@article {pmid41172978,
year = {2025},
author = {Sancho-Shimizu, V},
title = {VUS no more: Decoding inborn errors of immunity for clinical action.},
journal = {Cell},
volume = {188},
number = {22},
pages = {6105-6106},
doi = {10.1016/j.cell.2025.09.009},
pmid = {41172978},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Saturation genome editing meets functional phenotyping to turn sequencing ambiguity into actionable diagnoses.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems
Phenotype

@article {pmid41172903,
year = {2025},
author = {Zhang, J and Zhu, M and Yan, H and Qiao, J and Liu, Y},
title = {Split CRISPR/Cas systems: Pioneering solutions for molecular diagnostics challenges.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118177},
doi = {10.1016/j.bios.2025.118177},
pmid = {41172903},
issn = {1873-4235},
abstract = {Split CRISPR/Cas systems have recently emerged as revolutionary tools in molecular diagnostics, addressing the limitations of conventional CRISPR-based detection methods, such as low sensitivity and specificity for low-abundance targets and limited regulatory flexibility. This review highlights "split-activation" strategies that enhance analytical performance without requiring pre-amplification. It elaborates on two key approaches: split activator-mediated Cas systems for ultrasensitive RNA detection and split crRNA architectures for multiplex nucleic acid analysis. These innovations achieve significant improvements in sensitivity for femtomolar-level biomarkers while maintaining superior single-base discrimination. We examine their applications in clinical and on-site monitoring, analyze current challenges including background noise, multiplex capacity, and cost considerations. Future directions include developing customized Cas variants, nanomaterial-based amplification-free workflows, and integrated microfluidic platforms. This technology holds great promise for precision diagnostics, particularly in resource-limited settings.},
}

@article {pmid41171933,
year = {2025},
author = {Li, S and Vonesch, SC and Roy, KR and Tu, CS and Steudle, F and Nguyen, M and Jann, C and Steinmetz, LM},
title = {The editable landscape of the yeast genome reveals hotspots of structural variant formation.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eady9875},
pmid = {41171933},
issn = {2375-2548},
mesh = {*Genome, Fungal ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; *Genomic Structural Variation ; Whole Genome Sequencing ; INDEL Mutation ; },
abstract = {It is unclear how CRISPR editing outcomes vary across the genome and whether undesirable events such as structural variants (SVs) are predictable or preventable. To define a genome-wide map of editability, we performed whole-genome sequencing on 1875 budding yeast clones edited across 16 chromosomes by CRISPR-Cas9 and donor-templated repair. We found that unintended edits, including short indels and SVs, were enriched in specific genomic and sequence contexts. We developed a predictive model, SCORE (System for CRISPR Outcome and Risk Evaluation), which revealed 4.8% of the genome as SV prone, consisting of 562 SV hotspots. Donor repair-enhancing strategies suppressed SV formation in regions with moderate, but not high, predicted risk. Applying SCORE to the Sc2.0 synthetic yeast genome revealed a markedly altered SV landscape due to the removal of endogenous repetitive elements and the insertion of loxP sites. Our study provides the genome-scale map of SV hotspots after CRISPR editing and predictive and experimental tools to mitigate their formation.},
}

*Genome, Fungal
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Saccharomyces cerevisiae/genetics
*Genomic Structural Variation
Whole Genome Sequencing
INDEL Mutation

@article {pmid41171921,
year = {2025},
author = {Zhang, H and You, J and Zhou, H and Zhang, Z and Wu, H and Zhang, D and Pan, X and Zhang, W and Zhang, X and Rao, Z},
title = {The two-component nuclease-active KELShedu system confers broad antiphage activity via abortive infection.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eadv4747},
pmid = {41171921},
issn = {2375-2548},
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Bacteriophages/physiology ; *Escherichia coli Proteins/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Nucleotides/metabolism ; },
abstract = {Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX (Bacteriophage Exclusion), Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a double-stranded DNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of nucleotide triphosphates (NTPs) inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedu's activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to nonspecific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial antiphage mechanisms and lay the groundwork for applications in microbial engineering and therapeutic development.},
}

*Escherichia coli/virology/genetics/metabolism
*Bacteriophages/physiology
*Escherichia coli Proteins/metabolism/genetics
*DNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems
Nucleotides/metabolism

@article {pmid41171707,
year = {2025},
author = {Vadakkethil, AA and Panda, S and Mitra, A and Dash, M and Baig, MJ and Angadi, UB and Kumar, D and Jaiswal, S and Asif Iquebal, M and Molla, KA},
title = {CRISPR-GATE: a one-stop repository and guide to computational resources for genome editing experimentation.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {5},
pages = {},
pmid = {41171707},
issn = {1477-4054},
support = {//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; *Databases, Genetic ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR-Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
*Computational Biology/methods
*Software
*Databases, Genetic
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Internet

@article {pmid41171314,
year = {2025},
author = {Zaheer, A and AboQuella, NM and Wadan, AS and Saad, HA and Kumar, D and Panjwani, S and Rath, S and Ahmed, SI},
title = {CRISPR-based gene therapy for huntington's disease: current advances and future prospects.},
journal = {Neurogenetics},
volume = {26},
number = {1},
pages = {76},
pmid = {41171314},
issn = {1364-6753},
mesh = {*Huntington Disease/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Huntingtin Protein/genetics ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 technology offers transformative potential in treating Huntington's Disease (HD) by directly addressing its genetic root causes. This manuscript explores the pathophysiological mechanisms of HD, characterized by toxic mutant huntingtin (mHTT) protein resulting from expanded CAG repeats in the HTT gene, and the challenges posed by current therapeutic limitations. We comprehensively review the mechanisms of CRISPR-based therapeutic strategies, including excision of expanded repeats, allele-specific targeting, and epigenome editing, highlighting their efficacy in preclinical studies using animal models and human iPSCs. Delivery methods, such as viral and non-viral vectors, are analysed for their role in optimizing therapeutic outcomes while minimizing off-target effects and immune responses. Ethical and safety considerations, especially regarding precision and long-term impacts, are critically examined alongside emerging strategies to enhance specificity. With ongoing clinical trials and advancements in delivery systems, CRISPR technology represents a paradigm shift in addressing HD and broader neurodegenerative conditions. This review underscores the promise of gene editing in overcoming existing barriers and paving the way for transformative therapeutic approaches.},
}

*Huntington Disease/therapy/genetics
Humans
*Genetic Therapy/methods/trends
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
Huntingtin Protein/genetics
Disease Models, Animal

@article {pmid41170849,
year = {2025},
author = {Yu, T and Xie, J and Huang, X and Huang, J and Bao, G and Yuan, W and Gao, C and Liu, C and Hu, J and Yang, W and Li, G},
title = {BaeR and H-NS control CRISPR-Cas-mediated immunity and virulence in Acinetobacter baumannii.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0106725},
doi = {10.1128/msystems.01067-25},
pmid = {41170849},
issn = {2379-5077},
abstract = {Acinetobacter baumannii balances its remarkable ability to acquire antibiotic resistance genes via horizontal gene transfer (HGT) with the immune defense functions of its CRISPR-Cas system, forming a dynamic equilibrium governed by intricate transcriptional regulation. However, the regulatory mechanisms underlying the I-Fb CRISPR-Cas system in A. baumannii remain poorly understood. This study elucidated a multitiered regulatory axis mediated by BaeR and H-NS that coordinates immune defense and virulence expression in the I-Fb CRISPR-Cas system. Using DNA pull-down and electrophoretic mobility shift assay (EMSA), we demonstrated that H-NS directly binds AT-rich regions within the cas3 promoter, suppressing both interference activity and adaptive immunity of the I-Fb CRISPR-Cas system. Intriguingly, the two-component regulator BaeR controlled this suppression by positively regulating H-NS expression. The results revealed that Δcas3 mutants exhibited increased biofilm thickness, elevated the extracellular matrix component poly N-acetyl glucosamine (PNAG) production, upregulated pilus expression, and significantly enhanced epithelial cell adhesion. Strikingly, Δh-ns-cas3 and ΔbaeR-cas3 double-knockout strains showed no statistically significant differences in virulence phenotypes compared to the Δcas3 single mutants. These findings indicate CRISPR-Cas-mediated inhibition of biofilm formation is abolished upon cas3 deletion, thereby releasing the regulatory constraints imposed by BaeR and H-NS. This dysregulation leads to excessive biofilm and extracellular matrix component accumulation, ultimately amplifying bacterial colonization capacity and pathogenicity in host environments. This discovery reveals the dual regulatory roles of BaeR and H-NS in the A. baumannii I-Fb CRISPR-Cas system, mediating both immune defense and virulence modulation. These insights establish a theoretical foundation for novel antimicrobial strategies targeting CRISPR-Cas regulatory networks.IMPORTANCEA. baumannii, a leading cause of drug-resistant nosocomial infections, evolves antibiotic resistance through horizontal gene transfer (HGT) while employing CRISPR-Cas systems to limit foreign DNA invasion. This study reveals that the I-Fb CRISPR-Cas system, typically a defense mechanism, functions as a repressor of virulence traits in A. baumannii. We demonstrate that the transcriptional regulators H-NS and BaeR form a hierarchical axis suppressing Cas3 expression, thereby constraining biofilm formation and host adhesion. Strikingly, CRISPR-Cas deficiency enhances virulence, thickens biofilms, elevates PNAG production, and enhances epithelial colonization through escape from BaeR-/H-NS-mediated control. This work redefines CRISPR-Cas as a dual-function module balancing immune defense and pathogenicity, exposing the BaeR-H-NS-Cas3 axis as a druggable target for novel anti-infectives aimed at disrupting bacterial adaptive evolution.},
}

@article {pmid41072237,
year = {2026},
author = {Xin, M and Liu, J and Zhou, H and Bu, S and Hao, Z and Sun, H and Lu, J and Feng, X and Jiang, X and Wang, Q and Wan, J},
title = {An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification.},
journal = {Enzyme and microbial technology},
volume = {192},
number = {},
pages = {110759},
doi = {10.1016/j.enzmictec.2025.110759},
pmid = {41072237},
issn = {1879-0909},
mesh = {*Influenza A Virus, H1N1 Subtype/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Influenza, Human/diagnosis/virology ; SARS-CoV-2/isolation & purification/genetics ; RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/analysis/genetics ; DNA, Single-Stranded ; Bacterial Proteins ; },
abstract = {The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.},
}

*Influenza A Virus, H1N1 Subtype/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
Limit of Detection
Influenza, Human/diagnosis/virology
SARS-CoV-2/isolation & purification/genetics
RNA, Viral/analysis/genetics
Nucleic Acid Amplification Techniques/methods
DNA, Viral/analysis/genetics
DNA, Single-Stranded
Bacterial Proteins

@article {pmid40785075,
year = {2025},
author = {Zhu, L and Lin, Y and Yang, G and He, G and Pan, Z and Yu, G and Yao, J and Li, M and Zhou, L and Jiang, D},
title = {A Rapid On-Site Visualization Detection System for Fusarium oxysporum f. sp. cubense Tropical Race 4 Utilizing RPA-CRISPR/Cas12a.},
journal = {Plant disease},
volume = {109},
number = {10},
pages = {2062-2069},
doi = {10.1094/PDIS-11-24-2512-SR},
pmid = {40785075},
issn = {0191-2917},
mesh = {*Fusarium/genetics/isolation & purification ; *Plant Diseases/microbiology ; *Musa/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Fusarium wilt of banana, a disease with devastating impacts on banana plants, is primarily caused by a pathogenic fungus called Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). To facilitate control and interrupt the spread of Fusarium wilt of banana, we developed a rapid on-site visualization system for detecting Foc TR4 based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. Based on Foc TR4-specific sequences, primers, crRNA, and ssDNAs were designed. The detection system exhibited high specificity, with amplification signals observed exclusively in samples containing Foc TR4. Additionally, the system showed high sensitivity, with a limit of detection (LOD) of approximately 20 copies, and high efficiency, with detection results generated within 1 h from time of amplification. Notably, this method does not require large-scale instruments, making it a convenient and rapid approach. The detection system represents the first instance of on-site visualization of Foc TR4. This system can be employed for early detection of Foc TR4 and can provide a technical reference for the rapid detection and applications in the field of other pathogens.},
}

*Fusarium/genetics/isolation & purification
*Plant Diseases/microbiology
*Musa/microbiology
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40715781,
year = {2025},
author = {Zhang, H and Li, YC and Pang, D and Xie, C and Zhang, T and Li, Y and Li, Y and Jiang, ZY and Bu, GL and Liu, MM and Chen, YR and Fei, HX and Lin, RB and Wu, PH and Du, WT and Zhao, GX and Luo, YL and Han, P and Zhong, Q and Sun, C and Zeng, MS},
title = {Desmocollin 2 is a dominant entry receptor for Epstein-Barr virus infection of epithelial cells.},
journal = {Nature microbiology},
volume = {10},
number = {11},
pages = {2768-2780},
pmid = {40715781},
issn = {2058-5276},
mesh = {Animals ; Humans ; *Epithelial Cells/virology/metabolism ; *Virus Internalization ; *Desmocollins/metabolism/genetics ; *Herpesvirus 4, Human/physiology ; *Epstein-Barr Virus Infections/virology/metabolism ; Cell Line ; *Receptors, Virus/metabolism/genetics ; Cricetinae ; CRISPR-Cas Systems ; Viral Envelope Proteins/metabolism ; Membrane Glycoproteins/metabolism ; Viral Proteins ; Molecular Chaperones ; },
abstract = {Epstein-Barr virus (EBV) can infect B cells and epithelial cells, and cause lymphomas and various epithelial malignancies. During epithelial cell infection, EBV employs a complex combination of viral glycoproteins and host receptors. However, the exact mechanism and whether a dominant receptor exists remain unclear. Here we identify desmocollin 2 (DSC2) as a dominant EBV entry receptor for epithelial cell infection using CRISPR-Cas9 screening. Knockout of DSC2 reduced EBV infection in both nasopharyngeal and gastric epithelial cell lines, and infection was rescued when DSC2 expression was restored. Expression of human DSC2 in non-EBV-susceptible hamster cell lines enabled susceptibility to EBV. Furthermore, we found that DSC2 directly binds to EBV glycoprotein H/glycoprotein L through its extracellular domain, particularly the preEC-EC2 regions, which could be targeted by polyclonal antibodies, therefore blocking EBV infection in primary epithelial cells. DSC2 enabled virus entry independent of Ephrin receptor A2. These findings could aid development of currently unavailable animal models and support development of targeted therapies.},
}

Animals
Humans
*Epithelial Cells/virology/metabolism
*Virus Internalization
*Desmocollins/metabolism/genetics
*Herpesvirus 4, Human/physiology
*Epstein-Barr Virus Infections/virology/metabolism
Cell Line
*Receptors, Virus/metabolism/genetics
Cricetinae
CRISPR-Cas Systems
Viral Envelope Proteins/metabolism
Membrane Glycoproteins/metabolism
Viral Proteins
Molecular Chaperones

@article {pmid41170434,
year = {2025},
author = {Araújo, MRB and Dos Santos, LS and Prates, FD and Perini, HF and Silva, JS and Ramos, JN and Bokermann, S and Sacchi, CT and de Mattos Guaraldi, AL and Campos, KR and Cardoso, TDCS and Castro, DLC and Silva, MA and Sousa, MÂB and Vieira, VV and Santos, MBN and Camargo, CH and Andrade, BS and da Silva, MV and Sant'Anna, LO and Viana, MVC and Azevedo, V},
title = {Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1579154},
pmid = {41170434},
issn = {1664-302X},
abstract = {Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.},
}

@article {pmid41170433,
year = {2025},
author = {Zhang, S and Chu, M and Sun, X},
title = {The arms race in bacteria-phage interaction: deciphering bacteria defense and phage anti-defense mechanisms through metagenomics.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1687307},
pmid = {41170433},
issn = {1664-302X},
abstract = {Bacteriophages are viruses that specifically infect bacteria and co-evolve with their hosts through mutual interactions. They represent one of the most significant drivers of microbial diversity, influencing its evolution, generation, and maintenance. To counter bacteriophage infection, bacteria have developed sophisticated immune systems, including both passive adaptations, such as inhibiting phage adsorption and preventing DNA entry, and active defense systems such as restriction-modification systems and CRISPR-Cas systems. The ongoing arms race between bacteriophages and bacteria has left distinct evolutionary signatures in their genomic sequences. Advances in large-scale genomic and metagenomic sequencing technologies, coupled with bioinformatics approaches, have greatly enhanced our understanding of bacteria-phage interaction mechanisms, driving progress in bacteriophage biology. This review systematically analyses the diverse immune strategies bacteria employ against phage infection, elucidates the coordination and interrelationships among different anti-phage mechanisms, and highlights potential directions for future research.},
}

@article {pmid41168338,
year = {2025},
author = {Elsayed, EM and Stukenberg, D and Meier, D and Schmeck, B and Becker, A},
title = {RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1509},
pmid = {41168338},
issn = {2399-3642},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; },
mesh = {*Klebsiella pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Bacteriophage lambda/genetics ; *Genome, Bacterial ; },
abstract = {Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current Kp genome-editing tools are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system -Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing - as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.},
}

*Klebsiella pneumoniae/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Plasmids/genetics
*Bacteriophage lambda/genetics
*Genome, Bacterial

@article {pmid41167881,
year = {2025},
author = {Meng, X and Yue, Y and Huang, M and Duan, Z and Liu, K and Wu, L},
title = {DNAzyme-CRISPR driven dual-mode biosensor with nanozyme signal amplification for on-site Pb[2+] detection.},
journal = {Analytica chimica acta},
volume = {1379},
number = {},
pages = {344711},
doi = {10.1016/j.aca.2025.344711},
pmid = {41167881},
issn = {1873-4324},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; Electrochemical Techniques ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Limit of Detection ; Colorimetry ; Cerium/chemistry ; },
abstract = {BACKGROUND: Lead ion (Pb[2+]) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb[2+] detection.

RESULTS: To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO2 nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb[2+]-specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO2 to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb[2+] detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002-200 nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5-2000 nM; limit of detection: 0.47 nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5 % recovery in spiked corn, edible oil, beef and red wine samples with RSD <5 %. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb[2+].

SIGNIFICANCE: This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb[2+] in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.},
}

*Lead/analysis
*DNA, Catalytic/chemistry/metabolism
*Biosensing Techniques/methods
Electrochemical Techniques
*CRISPR-Cas Systems
Food Contamination/analysis
Limit of Detection
Colorimetry
Cerium/chemistry

@article {pmid41165985,
year = {2026},
author = {Bicknell, R and Koltunow, AMG},
title = {Pilosella: A Dicotyledonous Model for Studying Aposporous, Autonomous Apomixis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {209-220},
pmid = {41165985},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; Seeds/genetics/growth & development ; *Asteraceae/genetics/physiology/growth & development ; Ovule/genetics ; CRISPR-Cas Systems ; },
abstract = {Pilosella, a member of the Compositae, is a model system used to study the molecular genetics of aposporous apomixis. These plants are small, rapidly growing perennials that are easy to cultivate both in the greenhouse and in tissue culture. Apomixis in Pilosella occurs by apospory where mitotically derived embryo sacs arise adjacent to cells undergoing female gamete meiosis in the ovule. Seed initiation is autonomous, where both embryo and endosperm form without fertilization in the aposporous embryo sac. Apomixis is not fully penetrant in Pilosella. Instead, plants are facultatively apomictic, and apomixis can be easily scored through the simple decapitation of the immature capitulum bud. Natural sexual and facultatively apomictic forms are readily cross-compatible, facilitating comparative studies of inheritance and allele function. A wide range of experimental methods have been described for these plants, including histological techniques for studying the cytological aspects of apomixis, an efficient Agrobacterium-mediated transformation system, CRISPR/Cas9 mutagenesis, and mapping approaches that use deletion mutation and segregation in polyhaploid populations. Freely available online resources include a genome assembly, a molecular map based on cDNA markers and a transcriptome database. Collectively, these resources make Pilosella a highly tractable experimental system for studying the genetic control of native apomixis.},
}

*Apomixis/genetics
Seeds/genetics/growth & development
*Asteraceae/genetics/physiology/growth & development
Ovule/genetics
CRISPR-Cas Systems

@article {pmid41165983,
year = {2026},
author = {Radoeva, T and Rigola, D and Op den Camp, RHM and van Dijk, PJ and Underwood, CJ},
title = {Targeted Mutagenesis in Natural Apomicts.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {181-191},
pmid = {41165983},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; CRISPR-Cas Systems ; *Mutagenesis ; *Taraxacum/genetics ; Parthenogenesis/genetics ; Chromosome Mapping ; Genes, Plant ; },
abstract = {Apomixis-clonal reproduction through seeds-is an alternative reproductive strategy that takes place in less than 0.1% of plant species and has evolved independently in diverse plant lineages. To date, the genetic basis of apomixis has been unraveled genetically in only a few genera. The identification of causal apomixis genes is technically challenging, as apomictic species are typically polyploid and the genetic loci associated with apomixis are often in low-recombination regions limiting conventional fine-mapping. In triploid apomictic dandelion (Taraxacum officinale), after conventional genetic mapping, deletion mapping, and complete apomixis loci haplotype assembly, we made use of targeted mutagenesis using CRISPR/Cas9 technology to identify the Taraxacum officinale PARTHENOGENESIS (ToPAR) gene that is responsible for embryogenesis in the absence of fertilization. Here, we report the methods used to clone the ToPAR gene by targeted mutagenesis and we expect that the general principles could be applied in other systems to identify novel apomixis genes.},
}

*Apomixis/genetics
CRISPR-Cas Systems
*Mutagenesis
*Taraxacum/genetics
Parthenogenesis/genetics
Chromosome Mapping
Genes, Plant

@article {pmid41165418,
year = {2025},
author = {Kumar, U and Dwivedi, D and Das, U},
title = {Advancements in CRISPR-Mediated Multiplex Genome Editing: Transforming Plant Breeding for Crop Improvement and Polygenic Trait Engineering.},
journal = {Biotechnology journal},
volume = {20},
number = {11},
pages = {e70148},
doi = {10.1002/biot.70148},
pmid = {41165418},
issn = {1860-7314},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology/ ; 24J/01/00130//Council of Scientific and Industrial Research/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; AICE-JRF/SRF-KK04002406//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Multifactorial Inheritance/genetics ; Genome, Plant ; },
abstract = {With accelerating climate change and the urgent need to stack polygenic traits, multiplex CRISPR/Cas offers a scalable route to resilient crops-yet low editing efficiency and regeneration bottlenecks remain critical constraints. This review centers on multiplex strategies for polygenic trait engineering in plants, surveying compact nucleases (Cas9, Cas12, Cas13 and emerging ultra-compact variants), polycistronic gRNA platforms (tRNA-gRNA arrays, self-cleaving ribozymes, Csy4 processing), and delivery routes (Agrobacterium, biolistics, protoplast transfection, viral vectors). We highlight concrete outcomes-for example, targeted edits in PYL ABA-receptors increased rice grain yield by up to 31% in field tests-and applications from yield and disease resistance to abiotic-stress tolerance, nutrient biofortification and de novo domestication. Technical risks (off-targets, mosaicism, chromosomal rearrangements, transformability) are appraised alongside emerging fixes: compact/engineered nucleases, RNA-processing arrays, morphogenic regulators, and AI-driven sgRNA design integrated with multi-omics. By prioritizing multiplex approaches for polygenic trait stacking, the review argues that these tools are essential to accelerate precision breeding for climate-adapted agriculture.},
}

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

@article {pmid41163074,
year = {2025},
author = {Dashti, M and Mohammaddust Sarab, M and Shad, F and Dehnavi, S},
title = {CRISPR-mediated engineering of mesenchymal stromal/stem cells: a summary of recent progress in immunological applications for regenerative medicine and cancer therapy.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {592},
pmid = {41163074},
issn = {1757-6512},
mesh = {Humans ; *Mesenchymal Stem Cells/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology ; *Regenerative Medicine/methods ; Gene Editing ; Animals ; *Mesenchymal Stem Cell Transplantation ; },
abstract = {Mesenchymal stromal/stem cells (MSCs) have introduced as a cornerstone of regenerative medicine, owing to their immunomodulatory properties and therapeutic potential in autoimmune and inflammatory disorders. Although, their clinical application is often restricted due to immune rejection and heterogeneity in immunoregulatory responses. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has revolutionized MSC engineering, enabling precise genetic modifications to enhance their immunological efficacy. This review explores how CRISPR-mediated editing of MSCs can mitigate immunogenicity, amplify anti-inflammatory functions, and repurpose MSCs for targeted immunotherapy. Key strategies include knockout of β2-microglobulin to evade T-cell recognition, augmentation of anti-inflammatory mediators like interleukin (IL)-10 and TNF-alpha stimulated gene/protein 6 (TSG-6), and disruption of pro-inflammatory pathways such as toll-like receptor 4 (TLR4)/NF-κB. In addition, CRISPR-engineered MSCs demonstrate promise in reshaping tumor microenvironments and combating bacterial infections through enhanced innate immunity. Despite challenges including off-target effects and delivery optimization, CRISPR-tailored MSCs represent a transformative approach to overcoming immunological barriers, paving the way for universal, off-the-shelf therapies in rheumatoid arthritis, cancer, and beyond.},
}

Humans
*Mesenchymal Stem Cells/immunology/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Neoplasms/therapy/immunology
*Regenerative Medicine/methods
Gene Editing
Animals
*Mesenchymal Stem Cell Transplantation

@article {pmid41147512,
year = {2025},
author = {Mirzaei, F and Mosaffa Jahromi, A and Molavi, H and Kabelitz, D and Kalantar, K and Meri, S},
title = {Targeting RNA-Binding proteins Roquin-1 and Regnase-1 could enhance CAR-iPSC-derived macrophage immunotherapy for solid tumors: a perspective and challenges.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-7},
doi = {10.1080/15476286.2025.2581385},
pmid = {41147512},
issn = {1555-8584},
mesh = {Humans ; *Ribonucleases/genetics/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Macrophages/immunology/metabolism ; Tumor Microenvironment/immunology ; *RNA-Binding Proteins/genetics ; *Induced Pluripotent Stem Cells/cytology/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Ubiquitin-Protein Ligases/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Immunotherapy, Adoptive/methods ; Transcription Factors ; },
abstract = {Solid tumours present major treatment obstacles because of their immunosuppressive microenvironment and poor response to traditional chimeric antigen receptor (CAR)-based immunotherapies. Recent advances in cellular engineering have introduced CAR-macrophages derived from induced pluripotent stem cells (CAR-iMacs) as a promising approach to get around these obstacles. CAR-iMacs are designed to attack tumours, but their phenotypic plasticity can cause them to transform into M2-like macrophages in the tumour environment (TME), where they may instead suppress immune responses and promote tumour progression and metastasis. Roquin-1 and Regnase-1 are RNA-binding proteins that act as negative regulators of inflammatory genes that contribute to the phenotypic plasticity of macrophages. This perspective highlights a novel approach to augmenting anti-tumour responses of CAR-iMacs by simultaneously knocking out Roquin-1 and Regnase-1 via CRISPR-Cas9 gene editing. This approach drives a shift from an immunosuppressive M2-like state to an M1 state, promoting sustained pro-inflammatory signalling, boosting phagocytic and cytotoxic capabilities within the tumour microenvironment. Addressing a serious constraint in conventional adoptive cell therapies, this dual-targeting platform could provide a potent and scalable immunotherapeutic treatment for solid malignancies.},
}

Humans
*Ribonucleases/genetics/metabolism
*Neoplasms/therapy/immunology/genetics
*Macrophages/immunology/metabolism
Tumor Microenvironment/immunology
*RNA-Binding Proteins/genetics
*Induced Pluripotent Stem Cells/cytology/metabolism/immunology
Animals
*Immunotherapy/methods
Receptors, Chimeric Antigen/genetics/metabolism/immunology
*Ubiquitin-Protein Ligases/genetics
CRISPR-Cas Systems
Gene Editing
*Immunotherapy, Adoptive/methods
Transcription Factors

@article {pmid41037400,
year = {2025},
author = {Combredet, C and Ansel, M and Brunet, T},
title = {A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116345},
doi = {10.1016/j.celrep.2025.116345},
pmid = {41037400},
issn = {2211-1247},
mesh = {*Choanoflagellata/genetics/growth & development/metabolism ; *Signal Transduction ; *Gene Knockout Techniques/methods ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Choanoflagellates, the closest living relatives of animals, provide crucial insights into animal origins. The multicellular choanoflagellate Salpingoeca rosetta can be genetically modified, but existing knockout (KO) pipelines are time consuming and have variable efficiency. Here, we present a fast and robust KO method for S. rosetta. We use CRISPR-Cas9 to inactivate target genes by interrupting, or fully replacing, their coding sequence with a selectable antibiotic resistance cassette. We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble) and two homologs of Hippo pathway genes that control multicellular size in animals (warts and yorkie). Interestingly, warts-KO rosettes were consistently larger than their wild-type counterparts. RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous), suggesting that Hippo signaling regulates multicellular size in choanoflagellates by modulating matrix secretion. We discuss the potential of our method to accelerate choanoflagellate functional genetics.},
}

*Choanoflagellata/genetics/growth & development/metabolism
*Signal Transduction
*Gene Knockout Techniques/methods
*Protein Serine-Threonine Kinases/metabolism/genetics
Animals
CRISPR-Cas Systems/genetics

@article {pmid41026602,
year = {2025},
author = {Kalchschmidt, J and Kanno, T and Park, S and Dubois, WD and Zhao, Y and Trzaskoma, P and Thomas, CJ and Staudt, LM and O'Shea, JJ and Jung, S and Casellas, R},
title = {Biphasic control of the B cell transcriptome by mTORC1 and GSK3.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116361},
doi = {10.1016/j.celrep.2025.116361},
pmid = {41026602},
issn = {2211-1247},
mesh = {Humans ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *B-Lymphocytes/metabolism ; *Glycogen Synthase Kinase 3/metabolism/genetics ; *Transcriptome/genetics ; Lymphoma, B-Cell/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {A central question in immune regulation is how cells coordinate transcriptional responses to environmental cues. It remains unclear whether transcriptional regulation is controlled by isolated mechanism or integrated regulatory programs. Here, we develop a high-sensitivity, genome-wide CRISPR-Cas9 screening platform with 47 transcriptional reporters in human B cell lymphoma, identifying 4,440 regulators and 17,638 regulatory interactions. To enable the exploration of these networks, we establish B-LEARN, an interactive portal for data visualization and discovery. Our results reveal a large number of shared regulators across our 47 screens that act as context-dependent activators or repressors. Globally, we uncover a biphasic regulatory architecture in which mTORC1 and GSK3 exert opposing control over the B cell transcriptome. Notably, mTOR inhibition broadly activates key B cell genes, an effect antagonized by GSK3. Thus, B cell transcription is governed by an integrated, pathway-driven circuit, offering new targets to modulate gene expression in lymphoma and autoimmune disease.},
}

Humans
*Mechanistic Target of Rapamycin Complex 1/metabolism/genetics
*B-Lymphocytes/metabolism
*Glycogen Synthase Kinase 3/metabolism/genetics
*Transcriptome/genetics
Lymphoma, B-Cell/genetics
Cell Line, Tumor
CRISPR-Cas Systems/genetics

@article {pmid40693307,
year = {2025},
author = {Xia, Y and Guo, R and Lu, T and Jiang, S and You, K and Xia, X and Du, K and Kang, X},
title = {PagHB7/PagABF4-PagEPFL9 Module Regulates Stomatal Density and Drought Tolerance in Poplar.},
journal = {Plant biotechnology journal},
volume = {23},
number = {11},
pages = {4857-4871},
doi = {10.1111/pbi.70273},
pmid = {40693307},
issn = {1467-7652},
support = {2024M760220//the China Postdoctoral Science Foundation/ ; 2021YFD2200105//the National Key R&D Program of China during the 14th Five-year Plan Period/ ; },
mesh = {*Populus/genetics/physiology/metabolism ; *Plant Stomata/physiology/genetics ; Droughts ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Plants, Genetically Modified ; Stress, Physiological ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Epidermal patterning factor-like 9 (EPFL9) influences stomatal density and growth in poplar. There have been no reports on homeobox 7 (HB7) and ABRE binding factor 4 (ABF4) regulating stomatal density or drought tolerance by targeting EPFL9 in poplar. This study revealed that EPFL9 was specifically localised in guard cells in leaves and responded to drought stress. By constructing CRISPR/Cas9-mediated PagEPFL9 gene-edited lines, we found that epfl9 mutant plants showed significantly reduced stomatal density, inhibited growth and enhanced drought resistance. However, PagEPFL9 overexpression increased its drought stress sensitivity by increasing the stomatal density. PagHB7 was demonstrated to be an upstream regulator of PagEPFL9 by yeast one-hybrid screening library experiments, yeast one-hybrid experiments, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments. Yeast two-hybrid, bimolecular fluorescence complementation, split luciferase complementation assays, GST pull-down, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments further demonstrated that PagHB7 interacted with PagABF4 and that PagABF4 enhanced the inhibitory effect of PagHB7 on PagEPFL9. Knockout plants of PagHB7, a negative regulator of PagEPFL9, had a significantly increased stomatal density and reduced drought tolerance. Poplars overexpressing PagABF4 showed similar phenotypes to poplars knocking out PagEPFL9, with stomatal density significantly lower than that of WT, which may result in greater drought tolerance. Our study demonstrates that PagHB7 and PagABF4 interact with each other and regulate stomatal density by targeting PagEPFL9, thereby affecting drought resistance in poplar. This study provides new genetic resources for molecular design breeding of plant growth and drought tolerance.},
}

*Populus/genetics/physiology/metabolism
*Plant Stomata/physiology/genetics
Droughts
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant
*Transcription Factors/metabolism/genetics
Plants, Genetically Modified
Stress, Physiological
CRISPR-Cas Systems
Drought Resistance

@article {pmid41162659,
year = {2025},
author = {Campbell, IW and Basta, DW and Zingl, FG and Sullivan, EJ and Doranga, S and Waldor, MK},
title = {Anoxia activates CRISPR-Cas immunity in the mouse intestine.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41162659},
issn = {2058-5276},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; Investigator program//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here we find that the Citrobacter rodentium type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic environment of the mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologues, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats from foreign DNA within the intestinal microbiome.},
}

@article {pmid41162404,
year = {2025},
author = {Yan, X and Liu, B and Zhou, S and Fan, Y and Wei, S and Qiu, D and Xiang, H and Zhou, J and Mergny, JL and Monchaud, D and Ju, H and Zhou, J},
title = {A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04782},
pmid = {41162404},
issn = {1520-6882},
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.},
}

@article {pmid41162070,
year = {2025},
author = {Gautam, V and Jambagi, SR and Muthugounder, M},
title = {Genome editing of detoxification gene repertoires in insects using clustered regularly interspaced short palindromic repeats (CRISPR): A systematic review and meta-analysis.},
journal = {Pesticide biochemistry and physiology},
volume = {215},
number = {},
pages = {106687},
doi = {10.1016/j.pestbp.2025.106687},
pmid = {41162070},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Insecta/genetics ; *Insecticide Resistance/genetics ; Insecticides/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Inactivation, Metabolic/genetics ; },
abstract = {Decoding the molecular mechanisms underlying insect resistance to insecticides and host plant adaptation is essential for effective and sustainable Insecticide Resistance Management (IRM). Reverse genetic approaches targeting "target site resistance" can help control pests without harming pollinators and beneficial biocontrol agents. Understanding the role of mutations involved in xenobiotic resistance enables the judicious use of pesticides. CRISPR-based genome editing allows precise manipulation of detoxification genes, helping to decipher their roles in resistance development. This review provides a comprehensive overview of CRISPR-mediated genome editing in insect detoxification genes and their involvement in resistance mechanisms. In addition to synthesizing overall data trajectories, we present study-level effect sizes that highlight context-specific responses to gene editing, offering insights that can inform future experimental designs and functional validation studies.},
}

Animals
*Gene Editing
*Insecta/genetics
*Insecticide Resistance/genetics
Insecticides/pharmacology
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Inactivation, Metabolic/genetics

@article {pmid41161594,
year = {2025},
author = {Fu, H and Xu, W and Huang, M and Cong, Y},
title = {Molecular detection of Salmonella.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100659},
doi = {10.1016/j.jfp.2025.100659},
pmid = {41161594},
issn = {1944-9097},
abstract = {The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.},
}

@article {pmid41161575,
year = {2025},
author = {Verma, R and Das, G and Manjunathachar, H and Muwel, N and Choudhary, R and Kumar, S and Nath, S and Gattani, A and Gupta, V and Sharma, RK and Ajith, Y},
title = {CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111708},
doi = {10.1016/j.molbiopara.2025.111708},
pmid = {41161575},
issn = {1872-9428},
abstract = {Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.},
}

@article {pmid41161091,
year = {2025},
author = {Hong, SH and Kim, I and Lee, G and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.},
journal = {Biochemical and biophysical research communications},
volume = {789},
number = {},
pages = {152854},
doi = {10.1016/j.bbrc.2025.152854},
pmid = {41161091},
issn = {1090-2104},
abstract = {The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.},
}

@article {pmid41160700,
year = {2025},
author = {Elliott, SD and Ready, PJ and Wrinn, CM and Ma, Q and Edward, M and Niescier, RF and Escobar, I and Sun, J and Ganga, AK and McAtee, CK and Atiş, İS and Koleske, AJ and Bordey, A and Breslow, DK},
title = {A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eaeb7238},
pmid = {41160700},
issn = {2375-2548},
mesh = {*Cilia/metabolism/genetics/pathology ; Humans ; *Mutation ; *Malformations of Cortical Development/genetics/metabolism/pathology ; Cytoskeletal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; rhoA GTP-Binding Protein/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Armadillo Domain Proteins/genetics/metabolism ; Animals ; Signal Transduction ; Focal Cortical Dysplasia ; },
abstract = {Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein-coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.},
}

*Cilia/metabolism/genetics/pathology
Humans
*Mutation
*Malformations of Cortical Development/genetics/metabolism/pathology
Cytoskeletal Proteins/genetics/metabolism
*CRISPR-Cas Systems
rhoA GTP-Binding Protein/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Armadillo Domain Proteins/genetics/metabolism
Animals
Signal Transduction
Focal Cortical Dysplasia

@article {pmid40991409,
year = {2025},
author = {Smith, SL and Iwamoto, Y and Manimaran, A and Drubin, DG},
title = {Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo.},
journal = {Molecular biology of the cell},
volume = {36},
number = {11},
pages = {ar141},
doi = {10.1091/mbc.E25-06-0301},
pmid = {40991409},
issn = {1939-4586},
mesh = {Humans ; *Gene Editing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Endocytosis/genetics ; Cell Fusion/methods ; Actin Cytoskeleton/metabolism ; CRISPR-Cas Systems/genetics ; Clathrin/metabolism ; Lysosomes/metabolism ; },
abstract = {Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared with simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatial dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of CME and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes.},
}

Humans
*Gene Editing/methods
*Induced Pluripotent Stem Cells/metabolism/cytology
Endocytosis/genetics
Cell Fusion/methods
Actin Cytoskeleton/metabolism
CRISPR-Cas Systems/genetics
Clathrin/metabolism
Lysosomes/metabolism

@article {pmid40963020,
year = {2025},
author = {Chauhan, VP and Sharp, PA and Langer, R},
title = {Engineered prime editors with minimal genomic errors.},
journal = {Nature},
volume = {646},
number = {8087},
pages = {1254-1260},
pmid = {40963020},
issn = {1476-4687},
mesh = {*Gene Editing/methods ; *INDEL Mutation/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism/genetics ; Humans ; *Genome/genetics ; DNA/genetics/metabolism/chemistry ; *Genomics ; },
abstract = {Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends[1]. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors[2]. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.},
}

*Gene Editing/methods
*INDEL Mutation/genetics
CRISPR-Associated Protein 9/metabolism/genetics
*CRISPR-Cas Systems/genetics
Deoxyribonuclease I/metabolism/genetics
Humans
*Genome/genetics
DNA/genetics/metabolism/chemistry
*Genomics

@article {pmid41160667,
year = {2025},
author = {Du, X and Goh, PK and Ma, C and Coughlan, E and Greatorex, S and Porter, LH and Russ, B and Cummins, KD and Sek, K and Slaney, CY and Scott, AM and Oliaro, J and Neeson, PJ and Risbridger, GP and Taylor, RA and Trapani, JA and Turner, SJ and Darcy, PK and Wiede, F and Tiganis, T},
title = {Targeting PTPN2 enhances human CAR T cell efficacy and the development of long-term memory in mouse xenograft models.},
journal = {Science translational medicine},
volume = {17},
number = {822},
pages = {eadk0627},
doi = {10.1126/scitranslmed.adk0627},
pmid = {41160667},
issn = {1946-6242},
mesh = {Animals ; Humans ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists & inhibitors ; *Xenograft Model Antitumor Assays ; Mice ; *Immunologic Memory ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have been ineffective against solid tumors, where the hostile tumor microenvironment limits CAR T cell function and persistence. Protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor and cytokine signaling to maintain T cell tolerance. Here, we used CRISPR-Cas9 gene editing or an inhibitor to target PTPN2 in human CAR T cells specific for the Lewis Y (LeY) neoantigen, which is expressed in most epithelial tumors. Targeting PTPN2 increased CAR and cytokine signaling, including interferon signaling, and enhanced the antigen-induced expansion, activation, and cytotoxicity of anti-LeY CAR T cells in vitro and in vivo. The deletion of PTPN2 in CAR T cells repressed the growth of human tumor and patient-derived xenografts in mice, when compared with unedited CAR T cells, and prolonged mouse survival. The administration of inhibitor also enhanced the ability of α-LeY CAR T cells to repress tumor growth. Cellular indexing of transcriptomes and epitopes by sequencing analysis of splenic PTPN2-deficient CD8[+] CAR T cells in tumor-bearing mice revealed that PTPN2 deficiency favored the generation of CD45RA[+] CAR T cells expressing markers of long-lived stem cell memory (SCM) CAR T cells. Flow cytometric analysis reaffirmed that the deletion or inhibition of PTPN2 promoted the intratumoral accumulation of SCM CD8[+] CAR T cells and the overall persistence of CD8[+] CAR T cells. These data support the use of gene editing or small-molecule inhibitors targeting PTPN2 in human CAR T cells to treat solid tumors.},
}

Animals
Humans
*Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists & inhibitors
*Xenograft Model Antitumor Assays
Mice
*Immunologic Memory
*Receptors, Chimeric Antigen/metabolism/immunology
Immunotherapy, Adoptive
CRISPR-Cas Systems/genetics
Cell Line, Tumor
*T-Lymphocytes/immunology
CD8-Positive T-Lymphocytes/immunology

@article {pmid41160625,
year = {2025},
author = {Bish, LM and Fuss, JL and Panaccione, DG},
title = {Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334651},
doi = {10.1371/journal.pone.0334651},
pmid = {41160625},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Lysergic Acid/analogs & derivatives/metabolism ; *Amides/metabolism ; *Oxidoreductases/genetics/metabolism ; Peptide Synthases/genetics/metabolism ; Fungal Proteins/genetics/metabolism ; },
abstract = {Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Lysergic Acid/analogs & derivatives/metabolism
*Amides/metabolism
*Oxidoreductases/genetics/metabolism
Peptide Synthases/genetics/metabolism
Fungal Proteins/genetics/metabolism

@article {pmid41160062,
year = {2025},
author = {Yilmaz Çolak, Ç},
title = {Harnessing CRISPR technology for the diagnosis of Bordetella pertussis: advances and implications.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/17460913.2025.2581522},
pmid = {41160062},
issn = {1746-0921},
abstract = {Following the discovery of the prokaryotic adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins, this technology has revolutionized biotechnology as a multifaceted genome-editing tool with a wide range of applications. CRISPR technology has not only provided novel treatment options, especially for genetic diseases, but also transformed molecular diagnostic platforms. The specific, sensitive, and adaptable nature of the CRISPR-Cas systems has led to the development of innovative solutions for the detection of diseases, including viral and bacterial infections. This review provides an overview of the CRISPR-Cas systems and mainly focuses on the application of CRISPR-based assays for the detection of Bordetella pertussis, which is the main causative agent of a highly infectious disease, whooping cough. The review emphasizes the need for novel diagnostic tools for B. pertussis, along with highlighting some future perspectives, since its diagnosis can be challenging due to nonspecific early symptoms and interference from closely related Bordetella species. In this regard, CRISPR-based diagnostic platforms can offer a promising avenue for rapid and accurate detection of B. pertussis, helping the management of whooping cough.},
}

@article {pmid41159723,
year = {2025},
author = {Duan, Z and Yang, R and Lai, T and Jiang, W and Zhang, J and Chen, B and Liao, L},
title = {Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0177125},
doi = {10.1128/aem.01771-25},
pmid = {41159723},
issn = {1098-5336},
abstract = {Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.},
}

@article {pmid41157107,
year = {2025},
author = {Shpiliukova, K and Kachanov, A and Brezgin, S and Chulanov, V and Ivanov, A and Kostyushev, D and Kostyusheva, A},
title = {m[6]A RNA Modification: Technologies Behind Future Anti-Cancer Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {20},
pages = {},
doi = {10.3390/molecules30204091},
pmid = {41157107},
issn = {1420-3049},
support = {075-15-2025-519//Ministry of Science and Higher Education of the Russian Federation (Federal scientific and technical program for the development of genetic technologies for 2019-2030/ ; },
mesh = {Humans ; *Adenosine/analogs & derivatives/metabolism/genetics ; *Neoplasms/genetics/drug therapy/therapy/metabolism ; Methylation ; Animals ; Epigenesis, Genetic ; *Antineoplastic Agents/pharmacology/therapeutic use ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Methylation ; },
abstract = {N6-methyladenosine (m[6]A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m[6]A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m[6]A dynamics-marked by an imbalance in methylation and demethylation-can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m[6]A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m[6]A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m[6]A marks and altering m[6]A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m[6]A homeostasis or for strategically manipulating the m[6]A machinery for therapeutic benefit.},
}

Humans
*Adenosine/analogs & derivatives/metabolism/genetics
*Neoplasms/genetics/drug therapy/therapy/metabolism
Methylation
Animals
Epigenesis, Genetic
*Antineoplastic Agents/pharmacology/therapeutic use
*RNA/genetics/metabolism
*RNA Processing, Post-Transcriptional
RNA Methylation

@article {pmid41156655,
year = {2025},
author = {Peláez Sánchez, RG and González Restrepo, J and Pineda, S and Cuartas-López, AM and Martínez Garro, JM and Torres-Castro, M and Urrego, R and López-Rojas, LE and Salazar Florez, JE and Monroy, FP},
title = {Bioinformatic Identification of CRISPR-Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/pathogens14101044},
pmid = {41156655},
issn = {2076-0817},
mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/classification ; *Computational Biology/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Leptospirosis/microbiology ; },
abstract = {UNLABELLED: Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools.

METHODS: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST.

RESULTS: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems.

CONCLUSIONS: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.},
}

*CRISPR-Cas Systems/genetics
*Leptospira/genetics/classification
*Computational Biology/methods
Genome, Bacterial
Bacteriophages/genetics
Leptospirosis/microbiology

@article {pmid41155971,
year = {2025},
author = {Serrano, DR and Juste, F and Anaya, BJ and Ramirez, BI and Sánchez-Guirales, SA and Quispillo, JM and Hernandez, EM and Simon, JA and Trallero, JM and Serrano, C and Rawat, S and Lalatsa, A},
title = {Exosome-Based Drug Delivery: A Next-Generation Platform for Cancer, Infection, Neurological and Immunological Diseases, Gene Therapy and Regenerative Medicine.},
journal = {Pharmaceutics},
volume = {17},
number = {10},
pages = {},
doi = {10.3390/pharmaceutics17101336},
pmid = {41155971},
issn = {1999-4923},
support = {PID2024-156769OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; Innovation in Pharmacology, Nanotechnology, and personalized medicine by 3D printing//Universidad Complutense de Madrid/ ; },
abstract = {Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood-brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice.},
}

@article {pmid41155374,
year = {2025},
author = {Honjo, A and Yako, H and Miyamoto, Y and Yagi, M and Yamamoto, M and Nishi, A and Sakagami, H and Yamauchi, J},
title = {Knocking Down FRMD4A, a Factor Associated with the Brain Development Disorder and a Risk Factor for Alzheimer's Disease, Using RNA-Targeting CRISPR/Cas13 Reveals Its Role in Cell Morphogenesis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010083},
pmid = {41155374},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; Neurons/metabolism/cytology ; Mice ; *Morphogenesis/genetics ; *Cytoskeletal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Humans ; *Brain/metabolism ; Cell Line ; },
abstract = {Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer's disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired.},
}

*Alzheimer Disease/genetics/metabolism/pathology
Animals
*CRISPR-Cas Systems
Neurons/metabolism/cytology
Mice
*Morphogenesis/genetics
*Cytoskeletal Proteins/genetics/metabolism
*Membrane Proteins/genetics/metabolism
Gene Knockdown Techniques
Humans
*Brain/metabolism
Cell Line

@article {pmid41155368,
year = {2025},
author = {Hossain, MM and Sultana, F and Mostafa, M and Khan, I and Tran, LP and Mostofa, MG},
title = {Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010078},
pmid = {41155368},
issn = {1422-0067},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding ; *Plant Immunity/genetics ; *Genes, Plant ; Magnaporthe/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), poses a major threat to wheat (Triticum aestivum) cultivation, particularly in South America and Bangladesh. The rapid evolution and spread of the pathogen necessitate the development of durable and broad-spectrum resistance in wheat cultivars. This review summarizes current insights into the multi-layered defense mechanisms of wheat, encompassing resistance (R) genes, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) against MoT. The R-genes provide race-specific resistance through ETI, while both ETI and PTI are required to form integral layers of the plant immune system that synergistically reinforce host defense network. Recent advances in genomics, transcriptomics, and molecular breeding have facilitated the discovery and deployment of key R-genes and signaling components involved in PTI and ETI pathways. Integrating these immune strategies through gene pyramiding, marker-assisted selection (MAS), and genome editing offers a promising route towards enhanced and durable resistance in hosts. Harnessing and optimizing these multilayered immune systems will be pivotal to securing wheat productivity amid the growing threat of wheat blast.},
}

*Triticum/genetics/microbiology/immunology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
*Plant Breeding
*Plant Immunity/genetics
*Genes, Plant
Magnaporthe/pathogenicity
Host-Pathogen Interactions/genetics/immunology

@article {pmid41155240,
year = {2025},
author = {Carbone, F},
title = {Special Issue: Latest Research on Plant Genomics and Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms26209946},
pmid = {41155240},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *Genomics/methods ; *Plants/genetics ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; },
abstract = {Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].},
}

*Gene Editing/methods
*Genome, Plant
*Genomics/methods
*Plants/genetics
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing

@article {pmid41154642,
year = {2025},
author = {Zhang, H and Yang, Y and Yang, T and Cao, P and Yu, C and Liang, L and Liu, R and Chen, Z},
title = {Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.},
journal = {Biomolecules},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biom15101413},
pmid = {41154642},
issn = {2218-273X},
support = {2023YFC3402300//National Key R&D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//"Xingliao Talent Plan"project/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT25RC(3)019//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Mutation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.},
}

*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
*Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
*Endonucleases/genetics/metabolism
Mutation

@article {pmid41153450,
year = {2025},
author = {Nardon, E and Azzalini, E and Paladin, D and Boscarino, D and Bonin, S},
title = {CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101233},
pmid = {41153450},
issn = {2073-4425},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lyme Disease/diagnosis/microbiology/genetics ; *Borrelia burgdorferi Group/genetics/isolation & purification ; DNA, Bacterial/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Bacterial Outer Membrane Proteins/genetics ; Antigens, Surface ; Bacterial Vaccines ; Lipoproteins ; },
abstract = {BACKGROUND/OBJECTIVES: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.

METHODS: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.

RESULTS: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.

CONCLUSIONS: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.},
}

Humans
*CRISPR-Cas Systems/genetics
*Lyme Disease/diagnosis/microbiology/genetics
*Borrelia burgdorferi Group/genetics/isolation & purification
DNA, Bacterial/genetics
Real-Time Polymerase Chain Reaction/methods
Sensitivity and Specificity
Bacterial Outer Membrane Proteins/genetics
Antigens, Surface
Bacterial Vaccines
Lipoproteins

@article {pmid41153439,
year = {2025},
author = {Stunf Pukl, S},
title = {Genetic Therapy of Fuchs Endothelial Corneal Dystrophy: Where Are We? A Review.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101222},
pmid = {41153439},
issn = {2073-4425},
mesh = {*Fuchs' Endothelial Dystrophy/therapy/genetics ; Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Collagen Type VIII/genetics ; Mutation ; Transcription Factor 4/genetics ; Animals ; Gene Editing ; },
abstract = {OBJECTIVES: The incidence of Fuchs endothelial corneal dystrophy (FECD) is growing, and with it, the unmet need for a corneal transplant. Among alternative treatment modalities, only genetic therapy represents a causal therapy.

METHODS: Following the SNARA protocol, the PubMed and ClinicalTrials databases were searched using the keywords Fuchs endothelial corneal dystrophy, FECD, genetic therapy, and CRISPR-Cas9.

RESULTS: FECD is polyfactorial disease and mutations or polymorphisms in at least 15 different genes were connected to the disease. For the early-onset form of the disease, exclusive connection to mutations in COL8A2 was confirmed, while for the late-onset form, the most characteristic mutation is the expansion of the CTG18.1 triplet in the TCF4 gene, making these two possible targets. While the CRISPR-Cas9 approach represents the mainstay of genetic therapy development recently, the application of this method to FECD contains several obstacles, studied in preclinical settings. DT-168 and the Ad-Cas9-Col8a2gRNA molecules were developed for FECD treatment and preclinically tested, and phase I and II clinical studies for DT-168 are also already being performed.

CONCLUSIONS: The review of the literature proved that genetic therapy for FECD is at the level of preclinical research and that there are several specific challenges connected to the target genetic mutation as well as the delivery of possible treatment and duration of the effect. Further studies in the field might bring solutions in the future for alternative treatments for this common corneal disease.},
}

*Fuchs' Endothelial Dystrophy/therapy/genetics
Humans
*Genetic Therapy/methods
CRISPR-Cas Systems
Collagen Type VIII/genetics
Mutation
Transcription Factor 4/genetics
Animals
Gene Editing

@article {pmid41153385,
year = {2025},
author = {Boti, MA and Diamantopoulos, MA and Scorilas, A},
title = {RNA-Targeting Techniques: A Comparative Analysis of Modern Approaches for RNA Manipulation in Cancer Research and Therapeutics.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101168},
pmid = {41153385},
issn = {2073-4425},
mesh = {Humans ; *Neoplasms/genetics/therapy ; RNA Interference ; CRISPR-Cas Systems ; Oligonucleotides, Antisense/therapeutic use/genetics ; Animals ; *RNA/genetics ; },
abstract = {RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.},
}

Humans
*Neoplasms/genetics/therapy
RNA Interference
CRISPR-Cas Systems
Oligonucleotides, Antisense/therapeutic use/genetics
Animals
*RNA/genetics

@article {pmid41152294,
year = {2025},
author = {Nemoto, A and Imaizumi, K and Miya, F and Hiroi, Y and Yamada, M and Ideno, H and Saitoh, S and Kosaki, K and Okuno, H and Okano, H},
title = {Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9442},
pmid = {41152294},
issn = {2041-1723},
mesh = {*Prader-Willi Syndrome/genetics/therapy ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; CRISPR-Cas Systems ; *Epigenome/genetics ; Hypothalamus/metabolism ; Organoids/metabolism ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic ; Epigenome Editing ; },
abstract = {Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.},
}

*Prader-Willi Syndrome/genetics/therapy
Humans
*Genomic Imprinting/genetics
Induced Pluripotent Stem Cells/metabolism
*Gene Editing/methods
DNA Methylation/genetics
CRISPR-Cas Systems
*Epigenome/genetics
Hypothalamus/metabolism
Organoids/metabolism
Chromosomes, Human, Pair 15/genetics
Epigenesis, Genetic
Epigenome Editing

@article {pmid41152284,
year = {2025},
author = {Mishal, R and Meléndez-Zajgla, J and Rueda-Zarazúa, B and Labra-Barrios, ML and Castañón-Sánchez, CA and Uribe Carvajal, S and Padierna-Mota, L and Hernández-Hernández, JM and Leon-Avila, G and Pérez Rangel, A and Hernández-Martínez, E and Angeles-Morales, EB and Albalawi, IK and Luna-Arias, JP},
title = {RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {37578},
pmid = {41152284},
issn = {2045-2322},
support = {Ph.D. fellowship 465228//Consejo Nacional de Humanidades, Ciencia y Tecnologia, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; PhD. fellowship 752614//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *TATA-Box Binding Protein/genetics/metabolism ; *Mutation ; RNA-Seq ; Transcriptome ; Cell Proliferation/genetics ; Gene Expression Profiling ; Cell Movement/genetics ; },
abstract = {Breast cancer is the leading cause of death among women globally. Several genes have been found to be transcriptionally dysregulated in cancer, according to recent studies. TATA-box binding protein (TBP) and its two paralogs, TBPL1 and TBPL2, play roles in human transcription. The TBPL1 gene is implicated in colorectal carcinomas by suppressing the expression of miR-18a. However, its function in breast cancer remains undisclosed. TBPL1 is distantly related to TBP and possesses a 40% similarity with TBP's core domain. In the present study, we explored the potential role of the TBPL1 gene in transcriptome regulation by knocking out the TBPL1 gene through the CRISPR/Cas9 method. Following the knockout of the TBPL1 gene, we examined the gene transcription patterns and compared them to wild-type cell lines. We observed disparate signatures of upregulated and downregulated genes in wild-type and mutated conditions. Healthy breast MCF-12F, and T47D, SKBR3, and MDA-MB-231 breast cancer cell lines were assessed, as these cancer cells exhibit overexpression of the TBPL1 gene. Next-generation sequencing data revealed distinct marker genes regulated by the TBPL1 gene and their potential involvement in cell migration, proliferation, anti-apoptosis, and metastasis. Additionally, we also discovered novel lncRNAs implicated in the transcriptome analysis of the TBPL1 knocked-out gene. Our investigation indicated that this gene might affect varied stages of breast cancer cell lines' cellular properties, such as cell duplication, morphology, and growth. It might also contribute to tumor formation in more aggressive cell lines like MDA-MB-231 in vivo.},
}

Humans
*Breast Neoplasms/genetics/pathology
*CRISPR-Cas Systems
Female
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*TATA-Box Binding Protein/genetics/metabolism
*Mutation
RNA-Seq
Transcriptome
Cell Proliferation/genetics
Gene Expression Profiling
Cell Movement/genetics

@article {pmid41152217,
year = {2025},
author = {Song, Z and Guo, J and Fan, Z and Huang, S and Li, G and Zhao, Z and Chen, B and Huang, S and Zheng, W and Wei, Y and Chen, Y and Huang, X and Liu, J and Wu, L and Wang, X},
title = {Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9499},
pmid = {41152217},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Mice ; Male ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Protein Engineering ; },
abstract = {CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.},
}

*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics
*Cytosine/metabolism
Mice
Male
Humans
*CRISPR-Associated Proteins/metabolism/genetics
Protein Engineering

@article {pmid41151616,
year = {2025},
author = {Terhalle, E and Rademacher, J},
title = {[Nontuberculous Mycobacteria: Diagnostic Challenges and Individualized Therapeutic Approaches].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {150},
number = {22},
pages = {1360-1366},
doi = {10.1055/a-2502-6525},
pmid = {41151616},
issn = {1439-4413},
mesh = {Humans ; *Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy ; *Nontuberculous Mycobacteria/isolation & purification ; Anti-Bacterial Agents/therapeutic use ; Precision Medicine ; },
abstract = {Non-tuberculous mycobacteria (NTM) are increasingly recognized as clinically relevant pathogens, particularly in countries with a low tuberculosis incidence. Recent data from Denmark demonstrate a continuous annual rise in NTM-related pulmonary disease (NTM-PD) of 4.6% over 3 decades, with more than half of the isolates associated with true disease. Structural lung diseases such as bronchiectasis, prior tuberculosis, and chronic pulmonary conditions are major risk factors, alongside immunodeficiencies and immunosuppressive therapies. The diagnosis of NTM-PD requires a combination of clinical symptoms, radiological findings, and the microbiological confirmation. Novel diagnostic tools, such as anti-GPL IgA serology and a CRISPR-Cas-based cfDNA assay, show promise for differentiating colonization from disease and monitoring treatment response, but the sputum culture remains essential for species identification and drug susceptibility testing. Treatment is complex and species-specific, with macrolides forming the backbone of most regimens. Refractory cases, particularly those involving Mycobacterium abscessus, pose therapeutic challenges and often require multidisciplinary management. Inhaled liposomal amikacin (ALIS) has shown benefit in refractory MAC disease. Clinical decision-making must balance efficacy, tolerability, and long-term adherence, highlighting the need for individualized treatment strategies and regular monitoring. This review outlines current evidence and practical recommendations for clinicians managing NTM-PD.},
}

Humans
*Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy
*Nontuberculous Mycobacteria/isolation & purification
Anti-Bacterial Agents/therapeutic use
Precision Medicine

@article {pmid41149352,
year = {2025},
author = {Qin, Y and Xie, J and Zhen, S},
title = {CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149352},
issn = {2079-6374},
support = {22322409//National Natural Science Foundation of China/ ; 21974109//National Natural Science Foundation of China/ ; SWU-KF25011//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Hepatitis B virus/genetics ; *DNA, Viral/analysis ; *Fluorescence Polarization/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; Hepatitis B ; },
abstract = {Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5-9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.},
}

*Hepatitis B virus/genetics
*DNA, Viral/analysis
*Fluorescence Polarization/methods
Humans
*CRISPR-Cas Systems
*Biosensing Techniques
Limit of Detection
Hepatitis B