@article {pmid40157335,
year = {2025},
author = {Paranthaman, S and Uthaiah, CA and Md, S and Alkreathy, HM},
title = {Comprehensive strategies for constructing efficient CRISPR/Cas based cancer therapy: Target gene selection, sgRNA optimization, delivery methods and evaluation.},
journal = {Advances in colloid and interface science},
volume = {341},
number = {},
pages = {103497},
doi = {10.1016/j.cis.2025.103497},
pmid = {40157335},
issn = {1873-3727},
abstract = {Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.},
}

@article {pmid40157150,
year = {2025},
author = {Song, Y and Long, J and Wang, H and Tang, W and Yang, W and Zheng, Y and Yuan, R and Zhang, D and Gu, B and Nian, W},
title = {High-efficiency detection of APE1 using a defective PAM-driven CRISPR-Cas12a self-catalytic biosensor.},
journal = {Biosensors & bioelectronics},
volume = {279},
number = {},
pages = {117410},
doi = {10.1016/j.bios.2025.117410},
pmid = {40157150},
issn = {1873-4235},
abstract = {The trans-cleavage activity of the CRISPR-Cas system offers tremendous potential for developing highly sensitive and selective molecular diagnostic tools. However, conventional methods often face challenges such as limited catalytic efficiency of single Cas proteins and the necessity of complex multi-enzyme preamplification steps. To address these limitations, we present a novel defective PAM-mediated CRISPR-Cas12a self-catalytic signal amplification strategy, termed DEP-Cas-APE, for the rapid, sensitive, and specific detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity. This approach integrates defective PAM-modified DNA probes to synergize Cas12a trans-cleavage with self-catalytic circuit, achieving efficient signal transformation and amplification under isothermal, one-step conditions. We systematically investigated the influence of defective PAM sequences containing apurinic/apyrimidinic (AP) sites on Cas12a activation and validated the feasibility of the DEP-Cas-APE strategy in detecting APE1. Under optimized conditions, DEP-Cas-APE achieved a detection limit as low as 7.66 × 10[-8] U μL[-1] within 30 min using a simple isothermal reaction. Additionally, we developed a point-of-care testing (POCT) platform by integrating DEP-Cas-APE with a colorimetric assay based on gold nanoparticles (AuNPs), enabling portable, equipment-free detection. This sensitive and selective strategy successfully detected APE1 in complex biological samples, including serum from lung cancer patients, and demonstrated the ability to distinguish cancerous from normal samples. DEP-Cas-APE represents a robust and versatile platform for advancing CRISPR-Cas12a biosensing technologies, offering new opportunities for molecular diagnostics and clinical research.},
}

@article {pmid40156858,
year = {2025},
author = {Schmidt, T and Wiesbeck, M and Egert, L and Truong, TT and Danese, A and Voshagen, L and Imhof, S and Iraci Borgia, M and Deeksha, and Neuner, AM and Köferle, A and Geerlof, A and Santos Dias Mourão, A and Stricker, SH},
title = {Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf235},
pmid = {40156858},
issn = {1362-4962},
support = {//EpiCrossBorders/ ; //International Helmholtz-Edinburgh Research School for Epigenetics/ ; //Helmholtz Zentrum München/ ; STR 1385/5-1//DFG/ ; INST 86/2110-1//SFB/ ; //REGENERAR/ ; /CAPMC/CIHR/Canada ; //Natural Sciences and Engineering Research Council of Canada/ ; //Humanities Research Council of Canada/ ; },
mesh = {Humans ; *Ribonucleoproteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transcriptome ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; DNA/metabolism/genetics ; Promoter Regions, Genetic ; Transcriptional Activation ; },
abstract = {For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors.},
}

Humans
*Ribonucleoproteins/metabolism/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Transcriptome
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
DNA/metabolism/genetics
Promoter Regions, Genetic
Transcriptional Activation

@article {pmid40156857,
year = {2025},
author = {Vasquez, CA and Osgood, NRB and Zepeda, MU and Sandel, DK and Cowan, QT and Peiris, MN and Donoghue, DJ and Komor, AC},
title = {Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf037},
pmid = {40156857},
issn = {1362-4962},
support = {//University of California, San Diego/ ; 27502//Research Corporation for Science Advancement/ ; /HHMI/Howard Hughes Medical Institute/United States ; //National Academies of Sciences, Engineering, and Medicine/ ; //Cancer Cell Signaling and Communication Training Program/ ; T32 CA009523/GF/NIH HHS/United States ; //Molecular Biophysics Training/ ; },
mesh = {*DNA Glycosylases/genetics/metabolism ; Humans ; *Gene Editing/methods ; *DNA Repair/genetics ; *DNA Damage/genetics ; Mutation ; Oxidative Stress/genetics ; Colorectal Neoplasms/genetics ; Guanine/metabolism/analogs & derivatives ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Functional characterization of genetic variants has the potential to advance the field of precision medicine by enhancing the efficacy of current therapies and accelerating the development of new approaches to combat genetic diseases. MUTYH is a DNA repair enzyme that recognizes and repairs oxidatively damaged guanines [8-oxoguanine (8-oxoG)] mispaired with adenines (8-oxoG·A). While some mutations in the MUTYH gene are associated with colorectal cancer, most MUTYH variants identified in sequencing databases are classified as variants of uncertain significance. Convoluting clinical classification is the absence of data directly comparing homozygous versus heterozygous MUTYH mutations. In this study, we present the first effort to functionally characterize MUTYH variants using precision genome editing to generate heterozygous and homozygous isogenic cell lines. Using a MUTYH-specific lesion reporter in which we site-specifically incorporate an 8-oxoG·A lesion in a fluorescent protein gene, we measure endogenous MUTYH enzymatic activity and classify them as pathogenic or benign. Further, we modify this reporter to incorporate the MUTYH repair intermediate (8-oxoG across from an abasic site) and validate it with co-immunoprecipitation experiments to demonstrate its ability to characterize the mechanism by which MUTYH mutants are defective at DNA repair.},
}

*DNA Glycosylases/genetics/metabolism
Humans
*Gene Editing/methods
*DNA Repair/genetics
*DNA Damage/genetics
Mutation
Oxidative Stress/genetics
Colorectal Neoplasms/genetics
Guanine/metabolism/analogs & derivatives
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40156855,
year = {2025},
author = {Miceli, F and Bracaglia, S and Sorrentino, D and Porchetta, A and Ranallo, S and Ricci, F},
title = {MAIGRET: a CRISPR-based immunoassay that employs antibody-induced cell-free transcription of CRISPR guide RNA strands.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf238},
pmid = {40156855},
issn = {1362-4962},
support = {819160/ERC_/European Research Council/International ; 21965//Associazione Italiana per la Ricerca sul Cancro/ ; 2022ANCEK//Italian Ministry of University and Research/ ; 101165168/ERC_/European Research Council/International ; //Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Italian Ministry of University and Research/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Transcription, Genetic ; Immunoassay/methods ; CRISPR-Associated Proteins/metabolism ; Antibodies/immunology ; Humans ; Bacterial Proteins/genetics/metabolism ; Cell-Free System ; Endodeoxyribonucleases/metabolism ; Antigens/immunology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Here we report on the development of a CRISPR-based assay for the sensitive and specific detection of antibodies and antigens directly in complex sample matrices. The assay, called Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription (MAIGRET), is based on the use of a responsive synthetic DNA template that triggers the cell-free in vitro transcription of a guide RNA strand upon recognition of a specific target antibody. Such transcribed guide RNA activates the DNA collateral activity of the Cas12a enzyme, leading to the downstream cleavage of a fluorophore/quencher-labeled reporter and thus resulting in an increase in the measured fluorescence signal. We have used MAIGRET for the detection of six different antibodies with high sensitivity (detection limit in the picomolar range) and specificity (no signal in the presence of non-target antibodies). MAIGRET can also be adapted to a competitive approach for the detection of specific antigens. With MAIGRET, we significantly expand the scope and applicability of CRISPR-based sensing approaches to potentially enable the measurement of any molecular target for which an antibody is available.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Transcription, Genetic
Immunoassay/methods
CRISPR-Associated Proteins/metabolism
Antibodies/immunology
Humans
Bacterial Proteins/genetics/metabolism
Cell-Free System
Endodeoxyribonucleases/metabolism
Antigens/immunology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40156749,
year = {2025},
author = {Reķēna, A and Pals, K and Gavrilović, S and Lahtvee, PJ},
title = {The role of ATP citrate lyase, phosphoketolase, and malic enzyme in oleaginous Rhodotorula toruloides.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {77},
pmid = {40156749},
issn = {1432-0614},
mesh = {*Rhodotorula/genetics/enzymology/metabolism/growth & development ; *ATP Citrate (pro-S)-Lyase/metabolism/genetics ; *Gene Knockout Techniques ; *Malate Dehydrogenase/metabolism/genetics ; Xylose/metabolism ; Aldehyde-Lyases/metabolism/genetics ; Glucose/metabolism ; Lipid Metabolism ; CRISPR-Cas Systems ; Culture Media/chemistry ; Acetates/metabolism ; },
abstract = {Rhodotorula toruloides is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in R. toruloides IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in R. toruloides IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of R. toruloides. KEY POINTS: • ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880. • Lipid production and cell growth is are unchanged as a result of PK knockout. • Cytosolic malic enzyme does not play a significant role in lipogenesis.},
}

*Rhodotorula/genetics/enzymology/metabolism/growth & development
*ATP Citrate (pro-S)-Lyase/metabolism/genetics
*Gene Knockout Techniques
*Malate Dehydrogenase/metabolism/genetics
Xylose/metabolism
Aldehyde-Lyases/metabolism/genetics
Glucose/metabolism
Lipid Metabolism
CRISPR-Cas Systems
Culture Media/chemistry
Acetates/metabolism

@article {pmid40156192,
year = {2025},
author = {Georgiadis, C and Preece, R and Waseem Qasim, },
title = {Clinical development of allogeneic chimeric antigen receptor αβ-T Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.03.040},
pmid = {40156192},
issn = {1525-0024},
abstract = {Ready-made banks of allogeneic chimeric antigen receptor (CAR) T cells, produced to be available at the time of need, offer the prospect of accessible and cost-effective cellular therapies. Various strategies have been developed to overcome allogeneic barriers, drawing on cell engineering platforms including RNA interference, protein-based restriction and genome editing, including RNA-guided CRISPR-Cas and base editing tools. Alloreactivity and the risk of graft versus host disease from non-matched donor cells have been mitigated by disruption of αβ-T cell receptor expression on the surface of T cells, and stringent removal of any residual αβ-T cell populations. In addition, host mediated rejection has been tackled through a combination of augmented lymphodepletion and cell engineering strategies that have allowed infused cells to evade immune recognition or conferred resistance to lymphodepleting agents to promote persistence and expansion of effector populations. Early phase studies using 'off-the shelf' universal donor CAR T cells have been undertaken mainly in the context of blood malignancies, where emerging data of clinical responses have supported wider adoption and further applications. These developments offer the prospect of alternatives to current autologous approaches through the emerging application of genome engineering solutions to improve safety, persistence and function of universal donor products.},
}

@article {pmid40156040,
year = {2025},
author = {Zhang, Y and Zou, W and Zhou, Y and Chen, J and Hu, Y and Wu, F},
title = {Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {75},
pmid = {40156040},
issn = {1474-760X},
support = {YG2025QNB55//Research Fund of Medicine and Engineering of Shanghai Jiao Tong University/ ; 32271304//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Staphylococcus aureus/drug effects/genetics ; *DNA/metabolism ; *Streptococcus pyogenes/drug effects/enzymology ; Humans ; *Carbenoxolone/pharmacology ; CRISPR-Associated Protein 9/metabolism ; DNA Cleavage/drug effects ; Gene Editing ; HEK293 Cells ; },
abstract = {BACKGROUND: Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure.

RESULTS: Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid.

CONCLUSIONS: These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.},
}

Animals
Mice
*CRISPR-Cas Systems
*Staphylococcus aureus/drug effects/genetics
*DNA/metabolism
*Streptococcus pyogenes/drug effects/enzymology
Humans
*Carbenoxolone/pharmacology
CRISPR-Associated Protein 9/metabolism
DNA Cleavage/drug effects
Gene Editing
HEK293 Cells

@article {pmid40155371,
year = {2025},
author = {Chen, P and Wu, Y and Wang, H and Liu, H and Zhou, J and Chen, J and Lei, J and Sun, Z and Paek, C and Yin, L},
title = {Highly parallel profiling of the activities and specificities of Cas12a variants in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3022},
pmid = {40155371},
issn = {2041-1723},
support = {32371271//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32171210//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101196//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021TQ0253//China Postdoctoral Science Foundation/ ; 2022M712468//China Postdoctoral Science Foundation/ ; 2022M722473//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins/genetics/metabolism ; Deep Learning ; Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; },
abstract = {Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
HEK293 Cells
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins/genetics/metabolism
Deep Learning
Endodeoxyribonucleases/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
High-Throughput Nucleotide Sequencing

@article {pmid40154762,
year = {2025},
author = {Awan, MJA and Farooq, MA and Buzdar, MI and Zia, A and Ehsan, A and Waqas, MAB and Hensel, G and Amin, I and Mansoor, S},
title = {Advances in gene editing-led route for hybrid breeding in crops.},
journal = {Biotechnology advances},
volume = {81},
number = {},
pages = {108569},
doi = {10.1016/j.biotechadv.2025.108569},
pmid = {40154762},
issn = {1873-1899},
abstract = {With the global demand for sustainable agriculture on the rise, RNA-guided nuclease technology offers transformative applications in crop breeding. Traditional hybrid breeding methods, like three-line and two-line systems, are often labor-intensive, transgenic, and economically burdensome. While chemical mutagens facilitate these systems, they not only generate weak alleles but also produce strong alleles that induce permanent sterility through random mutagenesis. In contrast, RNA-guided nuclease system, such as clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein (Cas) system, facilitates more efficient hybrid production by inducing male sterility through targeted genome modifications in male sterility genes, such as MS8, MS10, MS26, and MS45 which allows precise manipulation of pollen development or pollen abortion in various crops. Moreover, this approach allows haploid induction for the rapid generation of recombinant and homozygous lines from hybrid parents by editing essential genes, like CENH3, MTL/NLD/PLA, and DMP, resulting in high-yield, transgene-free hybrids. Additionally, this system supports synthetic apomixis induction by employing the MiMe (Mitosis instead of Meiosis) strategy, coupled with parthenogenesis in hybrid plants, to create heterozygous lines and retain hybrid vigor in subsequent generations. RNA-guided nuclease-induced synthetic apomixis also enables genome stacking for autopolyploid progressive heterosis via clonal gamete production for trait maintenance to enhance crop adaptability without compromising yield. Additionally, CRISPR-Cas-mediated de novo domestication of wild relatives, along with recent advances to circumvent tissue culture- recalcitrance and -dependency through heterologous expression of morphogenic regulators, holds great promise for incorporating diversity-enriched germplasm into the breeding programs. These approaches aim to generate elite hybrids adapted to dynamic environments and address the anticipated challenges of food insecurity.},
}

@article {pmid40154215,
year = {2025},
author = {Zhang, Z and Ji, Q and Zhang, Z and Lyu, B and Li, P and Zhang, L and Chen, K and Fang, M and Song, J},
title = {Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases.},
journal = {Cancer genetics},
volume = {294-295},
number = {},
pages = {47-56},
doi = {10.1016/j.cancergen.2025.02.008},
pmid = {40154215},
issn = {2210-7762},
abstract = {BACKGROUND: Melanoma is a complex and often fatal disease, with NRAS being one of the most frequently mutated genes in this type of cancer. Liquid biopsies, specifically tests for circulating tumor DNA (ctDNA), represent a promising and less invasive approach to diagnosis. This study aims to develop an ultra-sensitive assay for detecting melanoma NRAS mutant ctDNA.

METHODS: To detect rare NRAS mutant ctDNA, we developed the NRAS PASEA assay by screening CRISPR-Cas proteins that recognize the PAM sequence 5'-TTN-3'. This method employs CRISPR-Cas proteins to continuously shear wild-type alleles during isothermal amplification, resulting in exponential amplification of mutant alleles to a detectable level by Sanger sequencing.

RESULTS: The developed NRAS Q61R/L/K mutation detection method can detect simulated ctDNA samples with mutant allele fractions (MAF) as low as 0.01 % with 30 mins of PASEA treatment. Notably, the NRAS Q61 K mutation was accurately identified by FnCas12a-based NRAS PASEA, even with the nucleotide at the "N" position in the PAM site "TTN." The method successfully detected ctDNA in patients with malignant melanoma. All patients (5/5) from 15 melanoma blood samples with NRAS Q61R (4/4) and NRAS Q61 K (1/1) mutations were accurately identified, with no false positives among patients with wildtype NRAS Q61.

CONCLUSION: Detecting ctDNA from peripheral blood samples is highly significant for melanomas in areas where imaging evaluation is challenging. Our assay demonstrated 100 % consistency with tumor tissue NGS, providing a new analytical strategy for companion diagnosis and dynamic assessment of therapeutic efficacy and disease progression in melanoma.},
}

@article {pmid40153308,
year = {2025},
author = {Hayhurst, M and Vink, JNA and Remerand, M and Gerth, ML},
title = {Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {3},
pages = {},
pmid = {40153308},
issn = {1465-2080},
mesh = {*Phytophthora/genetics/enzymology ; *Protoplasts/metabolism ; *Transformation, Genetic ; *Polyethylene Glycols/pharmacology ; Luminescent Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Plant Diseases/microbiology/parasitology ; Green Fluorescent Proteins/genetics/metabolism ; Plasmids/genetics ; Calcium Chloride/metabolism/pharmacology ; },
abstract = {Phytophthora species are eukaryotic plant pathogens that cause root rot and dieback diseases in thousands of plant species worldwide. Despite their significant economic and ecological impacts, fundamental molecular tools such as DNA transformation methods are not yet established for many Phytophthora species. In this study, we have established a PEG/calcium chloride (CaCl2)-mediated protoplast transformation method for Phytophthora agathidicida, the causal agent of kauri dieback disease. Adapting a protocol from Phytophthora sojae, we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for P. agathidicida protoplast formation, and the optimum pH of the recovery medium is 5. The media type did not significantly impact protoplast regeneration. Using this protocol, we generated transformants using three plasmids (i.e. pTdTomatoN, pYF2-PsNLS-Cas9-GFP and pYF2-PsNLS-Cas12a-GFP), which expressed fluorescent proteins and/or Cas nucleases. The transformants were unstable unless maintained under antibiotic selective pressure; however, under selection, fluorescence was maintained across multiple generations and life cycle stages, including the production of fluorescent zoospores from transformed mycelia. Notably, we observed the expression of GFP-tagged Cas nucleases, which is promising for future CRISPR-Cas genome editing applications. This study demonstrates that P. agathidicida is amenable to PEG/CaCl2-mediated protoplast transformation. Although the resulting transformants require antibiotic selective pressure to remain stable, this transient expression system can be valuable for applications such as cell tracking, chemotaxis studies and CRISPR-Cas genome editing. The protocol also provides a foundation for further optimization of transformation methods. It serves as a valuable tool for exploring the molecular biology of P. agathidicida and potentially other closely related Phytophthora species.},
}

*Phytophthora/genetics/enzymology
*Protoplasts/metabolism
*Transformation, Genetic
*Polyethylene Glycols/pharmacology
Luminescent Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing
Plant Diseases/microbiology/parasitology
Green Fluorescent Proteins/genetics/metabolism
Plasmids/genetics
Calcium Chloride/metabolism/pharmacology

@article {pmid40153053,
year = {2025},
author = {Kiraz, D and Özcan, A},
title = {Comparative genome analysis of 15 Streptococcus thermophilus strains isolated from Turkish traditional yogurt.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {4},
pages = {64},
pmid = {40153053},
issn = {1572-9699},
mesh = {*Yogurt/microbiology ; *Streptococcus thermophilus/genetics/classification/isolation & purification ; *Genome, Bacterial ; *Phylogeny ; Turkey ; Fermentation ; Whole Genome Sequencing ; Food Microbiology ; Genomics ; },
abstract = {Streptococcus thermophilus plays a pivotal role in yogurt fermentation, yet strains from traditional fermented products remain largely unexplored compared to their industrial counterparts. This study aimed to characterize the genomic diversity and functional potential of 15 S. thermophilus strains isolated from Turkish traditional yogurts, and to compare them with industrial strains. Through whole-genome sequencing and advanced bioinformatics analyses, we revealed distinct phylogenetic patterns and genetic features that differentiate these traditional strains from industrial isolates. The genomes (1.68-1.86 Mb) exhibited high genetic homogeneity (ANI > 98.69%) while maintaining significant functional diversity. Pan-genome analysis identified 1160 core genes and 5694 accessory genes, highlighting substantial genomic plasticity that enables niche adaptation. Our analysis uncovered several distinctive features: (1) unique phylogenetic clustering patterns based on both housekeeping genes and whole-genome SNPs, suggesting geographical isolation effects; (2) an extensive repertoire of carbohydrate-active enzymes (CAZymes), comprising 111 Glycoside Hydrolases, 227 Glycosyl Transferases, and 44 Carbohydrate Esterases and 13 Carbohydrate-Binding Modules, demonstrating sophisticated carbohydrate metabolism adaptation significantly enriched compared to industrial strains; (3) widespread GABA biosynthesis pathways in 8 strains, including complete gadB gene, indicating potential health-promoting properties; (4) multiple genomic islands containing genes for galactose utilization and stress response, suggesting specific adaptation to traditional fermentation environments; (5) diverse exopolysaccharide biosynthesis and bacteriocin gene clusters; and (6) widespread CRISPR-Cas systems with variable spacer content. Notably, we identified vanY glycopeptide resistance genes across all strains, with two strains additionally harboring vanT. These results reveal the genetic mechanisms behind S. thermophilus adaptation to traditional yogurt environments, offering valuable insights for developing starter cultures and preserving the unique qualities and potential health benefits of traditional dairy products.},
}

*Yogurt/microbiology
*Streptococcus thermophilus/genetics/classification/isolation & purification
*Genome, Bacterial
*Phylogeny
Turkey
Fermentation
Whole Genome Sequencing
Food Microbiology
Genomics

@article {pmid40152981,
year = {2025},
author = {Feng, B and Wang, Y and Zhang, X and Mu, T and Zhang, B and Li, Y and Zhang, H and Hua, W and Yuan, W and Li, H},
title = {Targeted mutagenesis and functional marker development of two Bna.TAC1s conferring novel rapeseed germplasm with compact architecture.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {4},
pages = {86},
pmid = {40152981},
issn = {1432-2242},
support = {32172028//National Natural Science Foundation of China/ ; U22A20477//National Natural Science Foundation of China/ ; },
mesh = {*Brassica napus/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Phenotype ; Genetic Markers ; Plant Breeding ; Mutagenesis ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Gene Editing ; Plants, Genetically Modified/genetics/growth & development ; Genes, Plant ; },
abstract = {Simultaneous disruption of two Bna.TAC1s, redundantly controlling the branch angle, generates a compact architecture in rapeseed, and two functional markers are developed to facilitate breeding rapeseed cultivars with compact architecture. Shoot branch angle is a key factor in determining the aerial plant architecture. A narrow branch angle can increase yields by facilitating mechanized harvest and high-density planting in rapeseed, a globally important oil crop. However, the available rapeseed varieties with narrow branch angle are very limited. In this study, two Bna.TAC1 members named BnaA5.TAC1 and BnaC4.TAC1 were found to have the four canonical domains of TAC1-like members, including domains I, II, III and IV in rapeseed. Each Bna.TAC1 exhibits dominant expression in the lateral branch with gradual dynamic response to light and encodes a protein localized in the plasma membrane. CRISPR/Cas9-mediated editing system was used to simultaneously knock out the two Bna.TAC1s to obtain two different Bna.tac1 double mutants, designed as CR-Bna.tac1-1 and CR-Bna.tac1-2. These two mutants displayed different degrees of compact architecture without affecting plant height and yield-related traits. The two Bna.TAC1s were also shown to play a redundant role in controlling branch angle by regulating the gravitropic response. In addition, we developed two specific gel-based functional markers in each Bna.TAC1 for the transgene-free mutant CR-Bna.tac1-1, which co-segregate with narrower branch angle and could help to identify the mutant alleles in a segregating population. We also found that the genomic variation of the two Bna.TAC1s is not associated with branch angle variation in the natural rapeseed population. Overall, these results reveal the key roles of Bna.TAC1s in regulation of rapeseed branch angle and provide a novel germplasm and functional markers for breeding superior varieties with compact architecture in rapeseed.},
}

*Brassica napus/genetics/growth & development
Plant Proteins/genetics/metabolism
Phenotype
Genetic Markers
Plant Breeding
Mutagenesis
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Gene Editing
Plants, Genetically Modified/genetics/growth & development
Genes, Plant

@article {pmid40152921,
year = {2025},
author = {Prithiviraj, S and Garcia Garcia, A and Linderfalk, K and Yiguang, B and Ferveur, S and Falck, LN and Subramaniam, A and Mohlin, S and Hidalgo Gil, D and Dupard, SJ and Zacharaki, D and Raina, DB and Bourgine, PE},
title = {Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40152921},
issn = {2050-084X},
support = {2019-01864_3//Vetenskapsrådet/ ; 948588/ERC_/European Research Council/International ; },
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism ; *Extracellular Matrix/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Rats ; Tissue Engineering/methods ; Core Binding Factor Alpha 1 Subunit/metabolism/genetics ; Cell Line ; Vascular Endothelial Growth Factor A/metabolism/genetics ; },
abstract = {Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.},
}

Humans
*Mesenchymal Stem Cells/metabolism
*Extracellular Matrix/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Rats
Tissue Engineering/methods
Core Binding Factor Alpha 1 Subunit/metabolism/genetics
Cell Line
Vascular Endothelial Growth Factor A/metabolism/genetics

@article {pmid40152384,
year = {2025},
author = {Fragale, N and Divvela, SSK and Williams-Ward, VC and Brand-Saberi, B},
title = {Loss of Atoh8 Affects Neurocranial and Axial Skeleton Development in Zebrafish.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {30},
number = {3},
pages = {26806},
doi = {10.31083/FBL26806},
pmid = {40152384},
issn = {2768-6698},
support = {G1001029//MRC/ ; MR/N021231/1//MRC/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Zebrafish Proteins/genetics/metabolism ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Bone Development/genetics ; Gene Knockout Techniques ; Skull/embryology/metabolism/growth & development ; },
abstract = {BACKGROUND: The basic helix-loop-helix (bHLH) transcription factor atonal homologue 8 (Atoh8) has been implicated in various developmental and physiological processes by means of transient knockdown and conditional knockout approaches in zebrafish, chick and mouse. Despite its demonstrated involvement in multiple tissues, the role of Atoh8 remains elusive in zebrafish. A recent permanent knockout study in zebrafish investigated the role of Atoh8 on the background of previous morpholino studies which demonstrated various developmental defects but could not find any of the morpholino-based effects in the mutant. In mice, a knockout study demonstrated involvement of the transcription factor in skeletal development, showing that disruption of the atoh8 gene results in reduction of skeletal size. We investigated a mutant fish line generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9)-technology for possible phenotypic effects on zebrafish skeletogenesis.

METHODS: Here, we present a CRISPR/Cas9-generated atoh8 permanent zebrafish mutant and investigate the phenotypic effects of the knockout on the developing zebrafish craniofacial and axial skeleton. We investigated the expression pattern of the gene in wildtype and conducted detailed morphometric analysis for a variety of bone and cartilage elements of the developing skeleton at 12 days post fertilisation (dpf) in zebrafish siblings from a heterozygous mating using detailed morphometric measurements and statistical analysis of the results.

RESULTS: Homozygous mutants are viable into late adulthood and show no overt morphological phenotype. Despite the prominent appearance of atoh8 signal in various embryonic and larval craniofacial and axial skeletal structures, detailed morphometric analysis revealed only subtle phenotypic effects of the mutation on skeletal development in zebrafish. We found the formation of the orbital cartilages of the developing neurocranium and the progress of chordacentra mineralisation to be negatively affected by loss of the transcription factor.

CONCLUSIONS: Despite the very subtle phenotypic effect of our mutation, we were able to show involvement of atoh8 in the skeletal development of zebrafish. We attribute the mild phenotype to a compensatory mechanism induced by nonsense-mediated degradation of messenger ribonucleic acid (mRNA) as suggested in the recent literature. The effect of atoh8-disruption on zebrafish skeletal development suggests that the loss of atoh8 cannot be compensated for at interfaces where more than one embryonic cell lineage contributes to bone and cartilage formation.},
}

Animals
*Zebrafish/genetics/embryology
*Zebrafish Proteins/genetics/metabolism
*Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Bone Development/genetics
Gene Knockout Techniques
Skull/embryology/metabolism/growth & development

@article {pmid40119192,
year = {2025},
author = {Rea, A and Santana-Hernández, S and Villanueva, J and Sanvicente-García, M and Cabo, M and Suarez-Olmos, J and Quimis, F and Qin, M and Llorens, E and Blasco-Benito, S and Torralba-Raga, L and Perez, L and Bhattarai, B and Alari-Pahissa, E and Georgoudaki, AM and Balaguer, F and Juan, M and Pardo, J and Celià-Terrassa, T and Rovira, A and Möker, N and Zhang, C and Colonna, M and Spanholtz, J and Malmberg, KJ and Montagut, C and Albanell, J and Güell, M and López-Botet, M and Muntasell, A},
title = {Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression.},
journal = {Nature immunology},
volume = {26},
number = {4},
pages = {582-594},
pmid = {40119192},
issn = {1529-2916},
support = {PI22/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; PI21/00002//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; ICI24/00041//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; FI23/00075//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; SGR863//Generalitat de Catalunya (Government of Catalonia)/ ; 2024PROD00089//Departament d'Innovació, Universitats i Empresa, Generalitat de Catalunya (Department of Innovation, Education and Enterprise, Government of Catalonia)/ ; 765104//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sklodowska-Curie Actions (H2020 Excellent Science - Marie Sklodowska-Curie Actions)/ ; 765104//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Smad4 Protein/metabolism/genetics ; *Activins/metabolism ; *Transforming Growth Factor beta/metabolism ; Mice ; Animals ; Cell Line, Tumor ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; Gene Knockout Techniques ; Cytotoxicity, Immunologic ; Neoplasms/immunology/therapy/metabolism ; Signal Transduction ; Cell Proliferation ; Xenograft Model Antitumor Assays ; },
abstract = {Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR-Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4[KO] NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4[KO] NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4[KO] conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*Smad4 Protein/metabolism/genetics
*Activins/metabolism
*Transforming Growth Factor beta/metabolism
Mice
Animals
Cell Line, Tumor
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods
Gene Knockout Techniques
Cytotoxicity, Immunologic
Neoplasms/immunology/therapy/metabolism
Signal Transduction
Cell Proliferation
Xenograft Model Antitumor Assays

@article {pmid40087529,
year = {2025},
author = {Kim, J and Nam, Y and Jeon, D and Choi, Y and Choi, S and Hong, CP and Kim, S and Jung, H and Park, N and Sohn, Y and Rim, YA and Ju, JH},
title = {Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {3},
pages = {686-699},
pmid = {40087529},
issn = {2092-6413},
support = {HI23C1234//Korea Health Industry Development Institute (KHIDI)/ ; 20024297//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; NRF-2023M3A9E4009811//Ministry of Education (Ministry of Education of the Republic of Korea)/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; HLA Antigens/genetics/immunology ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Hypoimmunogenic universal induced pluripotent stemn (iPS) cells were generated through the targeted disruption of key genes, including human leukocyte antigen (HLA)-A, HLA-B and HLA-DR alpha (DRA), using the CRISPR-Cas9 system. This approach aimed to minimize immune recognition and enhance the potential of iPS cells for allogeneic therapy. Heterozygous iPS cells were used for guide RNA design and validation to facilitate the knockout (KO) of the HLA-A, HLA-B and HLA-DRA genes. The electroporation of iPS cells using the selected guide RNAs enabled the generation of triple-KO iPS cells, followed by single-cell cloning for clone selection. Clone A7, an iPS cell with targeted KOs of the HLA-A, HLA-B and HLA-DRA genes, was identified as the final candidate. Messenger RNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4, sex-determining region Y box 2, Krüppel-like factor 4, Lin-28 homolog A and Nanog homeobox, while protein expression assays confirmed the presence of octamer-binding transcription factor 4, stage-specific embryonic antigen 4, Nanog homeobox and tumor rejection antigen 1-60. A karyotype examination revealed no anomalies, and three-germ layer differentiation assays confirmed the differentiation potential. After interferon gamma stimulation, the gene-corrected clone A7 lacked HLA-A, HLA-B and HLA-DR protein expression. Immunogenicity testing further confirmed the hypoimmunogenicity of clone A7, which was evidenced by the absence of proliferation in central memory T cells and effector memory T cells. In conclusion, clone A7, a triple-KO iPS cell clone that demonstrates immune evasion properties, retained its intrinsic iPS cell characteristics and exhibited no immunogenicity.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Gene Knockout Techniques
CRISPR-Cas Systems
Cell Differentiation/genetics
HLA Antigens/genetics/immunology
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39786412,
year = {2025},
author = {Uchida, K and Fuji, Y and Tabeta, H and Akashi, T and Hirai, MY},
title = {Omics-based identification of the broader effects of 2-hydroxyisoflavanone synthase gene editing on a gene regulatory network beyond isoflavonoid loss in soybean hairy roots.},
journal = {Plant & cell physiology},
volume = {66},
number = {3},
pages = {304-317},
doi = {10.1093/pcp/pcae151},
pmid = {39786412},
issn = {1471-9053},
support = {JP19K15821//Japan Society for the Promotion of Science/ ; JPJ012287//Cross-ministerial Strategic Innovation Promotion Program/ ; //soybean protein research/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Gene Editing/methods ; *Plant Roots/genetics/metabolism ; *Isoflavones/metabolism ; *Gene Regulatory Networks ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Flavonoids/metabolism ; },
abstract = {Soybean (Glycine max) is a leguminous crop cultivated worldwide that accumulates high levels of isoflavones. Although previous research has often focused on increasing the soybean isoflavone content because of the estrogen-like activity of dietary soy in humans, the rapidly increasing demand for soybean as a plant-based meat substitute has raised concerns about excessive isoflavone intake. Therefore, the production of isoflavone-free soybean has been anticipated. However, there have been no reports of an isoflavone-free soybean until now. Here, 2-hydroxyisoflavanone synthase (IFS), which is essential for isoflavone biosynthesis, was targeted for genome editing in soybean. A novel CRISPR/Cas9 system using Staphylococcus aureus Cas9 instead of the commonly used Streptococcus pyogenes Cas9 was established and customized. Through Agrobacterium rhizogenes-mediated transformation, IFS-edited hairy roots were generated in which all three IFS genes contained deletion mutations. Metabolome analyses of IFS-edited hairy roots revealed that isoflavone content significantly decreased, whereas levels of flavonoids, including a novel chalcone derivative, increased. A transcriptome analysis revealed changes in the expression levels of a large number of genes, including jasmonic acid-inducible genes. In addition, the functions of selected transcription factor genes (MYB14-L, GmbHLH112, and GmbHLH113), which were dramatically upregulated by IFS editing, were investigated by multiomics analyses of their over-expressing hairy root lines. They appear to be involved in flavonoid and triterpene saponin biosynthesis, salicylic acid metabolism, and central carbon metabolism. Overall, the results indicated that editing IFS genes caused the redirection of the metabolic flux from isoflavonoid biosynthesis to flavonoid accumulation, as well as dynamic changes in gene regulatory networks.},
}

*Glycine max/genetics/metabolism
*Gene Editing/methods
*Plant Roots/genetics/metabolism
*Isoflavones/metabolism
*Gene Regulatory Networks
*Gene Expression Regulation, Plant
CRISPR-Cas Systems
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Flavonoids/metabolism

@article {pmid40151969,
year = {2025},
author = {Tsubota, T and Takasu, Y and Yonemura, N and Sezutsu, H},
title = {Enhancements of the CRISPR-Cas System in the Silkworm Bombyx mori.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0089},
pmid = {40151969},
issn = {2573-1602},
abstract = {The silkworm (Bombyx mori) is a lepidopteran model insect that has been utilized for basic research and industrial applications. In this species, transcription activator-like effector nucleases (TALENs) have been found to function efficiently, and we previously developed a TALEN-mediated genome editing system for knockout and knock-in experiments using plasmids and single-stranded oligodeoxynucleotides (ssODNs) as donors. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing, especially for gene integration, remains limited. In this study, we attempted to improve CRISPR-Cas systems to expand the utility of genome editing in the silkworm. Codon optimization of Cas9 improved genome editing efficiency, and single-guide RNA utilization also resulted in a higher genome editing efficiency than crRNA/tracrRNA when Cas9 messenger RNA (mRNA) was used. CRISPR-Cas12a-mediated genome editing and targeted sequence integration using ssODNs were both successfully performed. Overall, our study provides a robust technical platform that can facilitate basic and applied silkworm studies.},
}

@article {pmid40151342,
year = {2025},
author = {Singh, A and Yasheshwar, and Kaushik, NK and Kala, D and Nagraik, R and Gupta, S and Kaushal, A and Walia, Y and Dhir, S and Noorani, MS},
title = {Conventional and cutting-edge advances in plant virus detection: emerging trends and techniques.},
journal = {3 Biotech},
volume = {15},
number = {4},
pages = {100},
pmid = {40151342},
issn = {2190-572X},
abstract = {Plant viruses pose a significant threat to global agriculture. For a long time, conventional methods including detection based on visual symptoms, host range investigations, electron microscopy, serological assays (e.g., ELISA, Western blotting), and nucleic acid-based techniques (PCR, RT-PCR) have been used for virus identification. With increased sensitivity, speed, and specificity, new technologies like loop-mediated isothermal amplification (LAMP), high-throughput sequencing (HTS), nanotechnology-based biosensors, and CRISPR diagnostics have completely changed the way plant viruses are detected. Recent advances in detection techniques integrate artificial intelligence (AI), machine learning (ML), and the Internet of Things (IoT) for real-time monitoring. Innovations like hyperspectral imaging, deep learning, and cloud-based IoT platforms further support disease identification and surveillance. Nanotechnology-based lateral flow assays and CRISPR-Cas systems provide rapid, field-deployable solutions. Despite these advancements, challenges such as sequence limitations, multiplexing constraints, and environmental concerns remain. Future research should focus on refining portable on-site diagnostic kits, optimizing nanotechnology applications, and enhancing global surveillance systems. Interdisciplinary collaboration across molecular biology, bioinformatics, and engineering is essential to developing scalable, cost-effective solutions for plant virus detection, ensuring agricultural sustainability and ecosystem protection.},
}

@article {pmid40151107,
year = {2025},
author = {Zou, G and Si, P and Wang, J and Yang, M and Chen, J and Liu, C and Luo, Z},
title = {Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation.},
journal = {ACS sensors},
volume = {10},
number = {3},
pages = {2073-2079},
doi = {10.1021/acssensors.4c03312},
pmid = {40151107},
issn = {2379-3694},
mesh = {*5-Methylcytosine/analogs & derivatives/analysis ; Humans ; *DNA Methylation ; *DNA/chemistry/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {5-Hydroxymethylcytosine (5hmC) plays a key role in the DNA demethylation process and serves as a stable epigenetic marker in the human genome which is closely associated with disease progression, particularly in diabetes, colorectal cancer, and liver cancer. However, convenient and sensitive methods for detecting and quantifying 5hmC in the genome are scarce, especially in complex biological environments. Herein, a novel attempt at hypersensitive quantitative detection of 5hmC was presented. A multifunctional photosensitive probe was therefore introduced for specific labeling, enrichment, and elution of 5hmC-DNA. Combining with isothermal assay leveraging rolling circle amplification and Cas12a for accurate recognition, we achieved quantitative detection of 5hmC DNA in trace amounts at a level of 11 fM. Global 5hmC was measured in various biological samples using as little as 10 ng of input DNA by a real-time PCR instrument. The reported approach imposed no sequence restrictions, demonstrating promising potential for detecting modified bases in trace amounts of nucleic acids within complex environments, such as blood, urine, and saliva samples.},
}

*5-Methylcytosine/analogs & derivatives/analysis
Humans
*DNA Methylation
*DNA/chemistry/genetics
CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Limit of Detection

@article {pmid40149851,
year = {2025},
author = {Pan, D and Mijit, M and Wang, H and Sun, C and Pingcuo, B and Yu, Z and Xiong, B and Tang, X},
title = {Rapid Genotyping of FecB Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform.},
journal = {Biomolecules},
volume = {15},
number = {3},
pages = {},
doi = {10.3390/biom15030315},
pmid = {40149851},
issn = {2218-273X},
support = {2022YFD1301102//Xiangfang Tang/ ; 2022BBF02020//Xiangfang Tang，Benhai Xiong/ ; Guike AA22068099//Xiangfang Tang/ ; 2004DA125184G2405//Hui Wang/ ; jc-cxgc-ias-09-1//Xiangfang Tang/ ; 2024-YWF-ZYSQ-10//Hui Wang/ ; CAAS-CSSAE-202402//Hui Wang/ ; },
mesh = {Animals ; Sheep/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Biosensing Techniques/methods ; Bone Morphogenetic Protein Receptors, Type I/genetics ; DNA/genetics ; Genotyping Techniques/methods ; Genotype ; },
abstract = {The A-to-G mutation (FecB) in the BMPR1B gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the FecB gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the FecB mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting FecB mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.},
}

Animals
Sheep/genetics
*CRISPR-Cas Systems/genetics
*Mutation
*Biosensing Techniques/methods
Bone Morphogenetic Protein Receptors, Type I/genetics
DNA/genetics
Genotyping Techniques/methods
Genotype

@article {pmid40148961,
year = {2025},
author = {Zheng, Y and Mo, Y and Yuan, Y and Su, T and Qi, Q},
title = {A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {74},
pmid = {40148961},
issn = {1475-2859},
support = {ZR2021QC021//Natural Science Foundation of Shandong Province/ ; tsqn202312029//Young Taishan Scholars Program of Shandong Province/ ; No. 32200081//National Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; *Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; N-Acetylneuraminic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge.

RESULTS: In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control.

CONCLUSION: We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.},
}

*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Plasmids/genetics
*Gene Expression Regulation, Bacterial
*Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics
Escherichia coli Proteins/genetics/metabolism
N-Acetylneuraminic Acid/metabolism
CRISPR-Cas Systems

@article {pmid40055023,
year = {2025},
author = {Ge, H and Feng, J and Huang, L and Luo, Z and Ling, H and Ma, L and Wang, M and Chen, H and Ren, L},
title = {Development of a highly sensitive, high-throughput and automated CRISPR-based device for the contamination-free pathogen detection.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117323},
doi = {10.1016/j.bios.2025.117323},
pmid = {40055023},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; Lab-On-A-Chip Devices ; Equipment Design ; Bacteria/isolation & purification/genetics ; Viruses/isolation & purification/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Rapid, portable, and contamination-resistant nucleic acid detection methods are necessary due to the threat posed by emerging viruses to public health and agricultural output. We establish CARE (CRISPR-associated airtight real-time electronic diagnostic device), a novel platform that combines CRISPR-Cas12a with a hermetically sealed microfluidic chip to overcome the limitations of present technologies, which struggle to balance sensitivity, multiplexing, and field applicability. By combining isothermal amplification and CRISPR detection within a hermetically sealed microfluidic chip, CARE eliminates the risk of nucleic acid aerosol contamination while enabling simultaneous high-throughput analysis of seven pathogens. The device is complemented by a user-friendly nucleic acid quantification App, enabling rapid and precise analysis. The RPA-CRISPR/Cas12a system demonstrates exceptional sensitivity, detecting as few as 1 copy μL[-1] (single-plex) and 10-10[2] copies μL[-1] (multiplexed), with real-sample performance matching gold-standard methods. CARE represents a significant advancement in CRISPR-based diagnostics, offering a robust, portable solution for on-site pathogen detection in food and agricultural applications.},
}

*Biosensing Techniques/instrumentation
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/instrumentation/methods
Humans
Lab-On-A-Chip Devices
Equipment Design
Bacteria/isolation & purification/genetics
Viruses/isolation & purification/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40054154,
year = {2025},
author = {Liu, S and Yang, Y and Li, X and Choi, JW and Guo, J and Luo, H and Li, C},
title = {Development of a single-tube RPA/CRISPR-cas12a detection platform for monkeypox virus.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117221},
doi = {10.1016/j.bios.2025.117221},
pmid = {40054154},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; Humans ; *Monkeypox virus/genetics/isolation & purification ; Limit of Detection ; Mpox, Monkeypox/diagnosis/virology ; Point-of-Care Testing ; CRISPR-Associated Proteins/genetics ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks primarily occurring in West and Central Africa. The recent global MPXV outbreak underscores the urgent need for effective detection methods. Currently, qPCR is considered the gold standard for MPXV detection; however, it requires specialized personnel and costly equipment. This study introduces a CRISPR-Cas12a-based detection system targeting the MPXV A27L gene, achieving a detection limit as low as 10 aM. This system exhibits high specificity, with no cross-reactivity with other orthopoxviruses, and delivers results in under 40 min. To support point-of-care testing (POCT), we developed a lateral flow assay (LFA) strip for easy result visualization. The detection system was validated using six different clinical sample types, revealing that herpes fluid and saliva are the most suitable sources. The findings of this study align with qPCR results. Additionally, we lyophilized the RPA and CRISPR reagents to improve transport, storage, and field deployment. In conclusion, this study presents a reliable molecular diagnostic approach for early MPXV detection and point-of-care testing, contributing to epidemic prevention and control.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods/instrumentation
Humans
*Monkeypox virus/genetics/isolation & purification
Limit of Detection
Mpox, Monkeypox/diagnosis/virology
Point-of-Care Testing
CRISPR-Associated Proteins/genetics
Animals
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39342962,
year = {2025},
author = {Zheng, L and Jiang, W and Zou, X and Song, L and Xu, X and Han, Y and Lian, H and Wu, X and Fang, X and Zhang, L},
title = {Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex.},
journal = {Plant disease},
volume = {109},
number = {3},
pages = {532-541},
doi = {10.1094/PDIS-07-24-1411-SR},
pmid = {39342962},
issn = {0191-2917},
mesh = {*Colletotrichum/genetics/isolation & purification ; *Fragaria/microbiology ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; Thiophenes/chemistry/pharmacology ; Seedlings/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides species complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of the CGSC in strawberry seedlings. This method targets the β-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA. This method shows high sensitivity (10 copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1 to 2 min). Furthermore, the Cas12aVIP assay successfully detected the CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for the CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting the CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.},
}

*Colletotrichum/genetics/isolation & purification
*Fragaria/microbiology
*Plant Diseases/microbiology
CRISPR-Cas Systems
Thiophenes/chemistry/pharmacology
Seedlings/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40148327,
year = {2025},
author = {Zhang, X and Li, M and Chen, K and Liu, Y and Liu, J and Wang, J and Huang, H and Zhang, Y and Huang, T and Ma, S and Liao, K and Zhou, J and Wang, M and Lin, Y and Rong, Z},
title = {Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3016},
pmid = {40148327},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Adenine/metabolism/chemistry ; Mice ; Humans ; CRISPR-Associated Proteins/metabolism/genetics ; HEK293 Cells ; Transcriptional Activation ; Liver/metabolism ; Genetic Therapy/methods ; },
abstract = {CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9-19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3-3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2-2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Adenine/metabolism/chemistry
Mice
Humans
CRISPR-Associated Proteins/metabolism/genetics
HEK293 Cells
Transcriptional Activation
Liver/metabolism
Genetic Therapy/methods

@article {pmid40147084,
year = {2025},
author = {Chen, X and Zhao, D and Yu, C and Wei, J and Zhou, G},
title = {A novel photosensitive nanoprobe combined with CRISPR/Cas12a for dual signal amplification detection of ANGPTL2.},
journal = {Talanta},
volume = {292},
number = {},
pages = {128010},
doi = {10.1016/j.talanta.2025.128010},
pmid = {40147084},
issn = {1873-3573},
abstract = {The detection of specific protein biomarkers holds significant potential for the early diagnosis of colorectal cancer (CRC). However, the accurate quantification of low-abundance proteins in serum presents a major challenge due to factors such as limited sensitivity and the complexity of the required methodologies. In this work, we established a universal CRISPR/Cas biosensing platform by integrating novel photosensitive nanoprobes (DA/PL@Cu NPs) and CRISPR/Cas12 system (DPC-Cas) for the highly sensitive, specific and user-friendly detection of angiopoietin-like protein 2 (ANGPTL2). The DA/PL@Cu NPs serve as a critical component in the transduction of protein recognition information into nucleic acid amplification events to produce Cas12a activators. The DPC-Cas biosensor integrates DA/PL@Cu NPs-assisted amplification with Cas12a self-amplification, enabling ultrasensitive detection of ANGPTL2 at concentrations as low as 20.00 pg/mL. The proposed DPC-Cas biosensor successfully detected ANGPTL2 in serum, demonstrating significant potential for the early diagnosis of CRC.},
}

@article {pmid40146761,
year = {2025},
author = {Winiarczyk, D and Khodadadi, H and Leszczyński, P and Taniguchi, H},
title = {A simple validation and screening method for CRISPR/Cas9-mediated gene editing in mouse embryos to facilitate genetically modified mice production.},
journal = {PloS one},
volume = {20},
number = {3},
pages = {e0312722},
pmid = {40146761},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Embryo, Mammalian/metabolism ; Female ; Blastocyst/metabolism ; Mice, Transgenic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a genome engineering method for generating site-specific editing in target genes in a variety of species. It is a common tool for generating mouse models of different diseases. However, detecting target modifications in mouse embryos can be time-consuming and expensive. Accordingly, developing a screening method to confirm gene modification may be useful. We propose herein an evaluation method (cleavage assay - CA) for CRISPR/Cas9-mediated gene editing in preimplantation mouse embryos that allows us to detect mutants efficiently and later on initiate in vivo production without the extensive number of samples needing to be sent for Sanger sequencing and animal usage. Our method is based on the inability of the RNP complex to recognize the target sequence after CRISPR-mediated genome editing due to modification of the target locus. It allows us to establish gene edited mice in a user-friendly fashion with a limited number of mice usage by confirming each step of CRISPR-mediated gene editing of mouse embryos and, therefore, can be considered as a supportive tool to existing procedures for verification of successful CRISPR/Cas9-mediated gene alterations in mouse embryos and further mutant production.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Embryo, Mammalian/metabolism
Female
Blastocyst/metabolism
Mice, Transgenic
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40146517,
year = {2025},
author = {Tripathi, JN and Tripathi, L},
title = {Agrobacterium tumefaciens-Mediated Genome Editing in Banana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {143-153},
pmid = {40146517},
issn = {1940-6029},
mesh = {*Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; *Musa/genetics/microbiology ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; *Transformation, Genetic ; Genome, Plant ; Plasmids/genetics ; },
abstract = {This protocol outlines the Agrobacterium tumefaciens-mediated transformation process for genome editing in banana (Musa spp.). As a crop of significant agricultural and economic importance globally, improving banana cultivars is crucial for addressing challenges such as disease resistance, climate resilience and yield enhancement. The procedure presented here involves the establishment of embryogenic cell suspensions (ECSs) from immature male flowers, followed by co-cultivation with Agrobacterium containing the desired gene construct. Key steps include callus induction, ECS generation and maintenance, transformation using plasmid construct containing genome editing reagents such as CRISPR/Cas9, and regeneration. The method ensures the editing of target genes in the banana genome, facilitating genetic improvements.},
}

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

@article {pmid40146515,
year = {2025},
author = {Arruabarrena, A and Vidal, S},
title = {Agrobacterium-Mediated Transformation for Gene Editing Tomato Elite Breeding Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {121-131},
pmid = {40146515},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; *Plant Breeding/methods ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; Agrobacterium/genetics ; Agrobacterium tumefaciens/genetics ; },
abstract = {Genome editing success in crop species is largely dependent on the availability of highly efficient plant transformation protocols. Tomato (Solanum lycopersicum) was the first dicotyledonous crop to be successfully mutagenized using CRISPR-Cas9. Despite many efforts, no standardized, simple protocol is available for non-model tomato genotypes. With the increasing availability of gene editing tools, the transformation of elite tomato breeding lines has gained importance because mutant variants can be easily incorporated into breeding programs. This chapter describes a protocol for transforming and gene editing in elite tomato breeding lines, reaching 3.6% transformation efficiency.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*Transformation, Genetic
*Plant Breeding/methods
*CRISPR-Cas Systems
*Plants, Genetically Modified/genetics
Agrobacterium/genetics
Agrobacterium tumefaciens/genetics

@article {pmid40146510,
year = {2025},
author = {Castillo Castro, FM and Payacán Ortiz, C},
title = {Agrobacterium-Mediated Transformation for Commercial Wheat Varieties.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {61-70},
pmid = {40146510},
issn = {1940-6029},
mesh = {*Triticum/genetics/growth & development/microbiology ; *Transformation, Genetic ; *Agrobacterium tumefaciens/genetics ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plant Breeding/methods ; },
abstract = {Wheat is a crucial crop for global food security, and new breeding techniques face limitations due to low regeneration rates and a lack of transformable genotypes. We follow and adapt a robust Agrobacterium tumefaciens-mediated transformation system in spring commercial wheat varieties from Chile and Argentina grown in speed breeding conditions. By incorporating the developmental gene fusion GRF4-GIF1, we achieved successful regeneration of plantlets in different wheat varieties through CRISPR/Cas9-based gene editing. This advancement reduces genotype dependency, allowing broader use of genome-editing tools in commercial wheat varieties. In addition to delving into technical complexities, this contribution aims to advance fundamental understanding and practical applications in wheat genetics, serving as a valuable resource for researchers expanding their expertise.},
}

*Triticum/genetics/growth & development/microbiology
*Transformation, Genetic
*Agrobacterium tumefaciens/genetics
*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Plant Breeding/methods

@article {pmid40146509,
year = {2025},
author = {Faion-Molina, M and Molina-Risco, MD and Bellinatti-Della Gracia, MD and Ibarra, O and Kim, B and Septiningsih, EM and Thomson, MJ},
title = {Agrobacterium tumefaciens-Mediated Plant Transformation and Gene Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {45-59},
pmid = {40146509},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; *CRISPR-Cas Systems ; *Agrobacterium tumefaciens/genetics ; *Plants, Genetically Modified/genetics ; Oxidoreductases ; },
abstract = {Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas9-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent most of the rice production in the USA and South America, along with some regions in Asia. This chapter describes a protocol for CRISPR/Cas9 gene editing using Agrobacterium-mediated transformation for the tropical japonica rice cultivar Presidio leading to knock-out mutations in the phytoene desaturase (PDS) gene.},
}

*Oryza/genetics
*Gene Editing/methods
*Transformation, Genetic
*CRISPR-Cas Systems
*Agrobacterium tumefaciens/genetics
*Plants, Genetically Modified/genetics
Oxidoreductases

@article {pmid40146284,
year = {2025},
author = {Kaniganti, S and Palakolanu, SR and Thiombiano, B and Damarasingh, J and Bommineni, PR and Che, P and Sharma, KK and Jones, T and Bouwmeester, H and Bhatnagar-Mathur, P},
title = {Developing Striga resistance in sorghum by modulating host cues through CRISPR/Cas9 gene editing.},
journal = {Plant cell reports},
volume = {44},
number = {4},
pages = {90},
pmid = {40146284},
issn = {1432-203X},
support = {DBT/2017/ICRISAT/973//Department of Biotechnology, Minstry of Science and Technology, India (IN)/ ; },
mesh = {*Sorghum/genetics/parasitology ; *CRISPR-Cas Systems ; *Striga/physiology ; *Gene Editing/methods ; *Plant Diseases/genetics/parasitology/immunology ; *Disease Resistance/genetics ; *Plants, Genetically Modified ; Mutation ; Lactones/metabolism ; Plant Proteins/genetics/metabolism ; Plant Weeds/genetics ; },
abstract = {High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E0 lines in the E1 generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.},
}

*Sorghum/genetics/parasitology
*CRISPR-Cas Systems
*Striga/physiology
*Gene Editing/methods
*Plant Diseases/genetics/parasitology/immunology
*Disease Resistance/genetics
*Plants, Genetically Modified
Mutation
Lactones/metabolism
Plant Proteins/genetics/metabolism
Plant Weeds/genetics

@article {pmid40144376,
year = {2025},
author = {Smirnov, AV and Korablev, AN and Serova, IA and Yunusova, AM and Muravyova, AA and Valeev, ES and Battulin, NR},
title = {Studying concatenation of the Cas9-cleaved transgenes using barcodes.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {1},
pages = {26-34},
doi = {10.18699/vjgb-25-04},
pmid = {40144376},
issn = {2500-0462},
abstract = {In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9- linearized transgenes (RNP- experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP- experiment, six positive embryos were identified, four of which exhibited lowcopy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2-4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/ Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.},
}

@article {pmid40143364,
year = {2025},
author = {Chakraborty, M and Nielsen, L and Nash, D and Nissimov, JI and Charles, TC and Aucoin, MG},
title = {Adapting Next-Generation Sequencing to in Process CRISPR-Cas9 Genome Editing of Recombinant AcMNPV Vectors: From Shotgun to Tiled-Amplicon Sequencing.},
journal = {Viruses},
volume = {17},
number = {3},
pages = {},
pmid = {40143364},
issn = {1999-4915},
support = {RGPIN 355513-2017//Natural Sciences and Engineering Research Council/ ; RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Nucleopolyhedroviruses/genetics ; *Genetic Vectors/genetics ; Animals ; *High-Throughput Nucleotide Sequencing ; Sf9 Cells ; *Genome, Viral ; Spodoptera/genetics/virology ; Cell Line ; },
abstract = {The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. AcMNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant AcMNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Nucleopolyhedroviruses/genetics
*Genetic Vectors/genetics
Animals
*High-Throughput Nucleotide Sequencing
Sf9 Cells
*Genome, Viral
Spodoptera/genetics/virology
Cell Line

@article {pmid40143350,
year = {2025},
author = {Wu, H and Wang, Z and Zhang, Y and Hu, L and Yang, J and Zhang, C and Lou, M and Pi, N and Wang, Q and Fan, S and Huang, Z},
title = {A New Human SCARB2 Knock-In Mouse Model for Studying Coxsackievirus A16 and Its Neurotoxicity.},
journal = {Viruses},
volume = {17},
number = {3},
pages = {},
pmid = {40143350},
issn = {1999-4915},
support = {NO.202105AD160018//Technology Innovation Talents Project of Yunnan Province/ ; NO. 2021-I2M-1-043//CAMS Innovation Fund for Medical Sciences (CIFMS)/ ; U2202214//Joint Funds of the National Natural Science Foundation of China/ ; 2023ZD040630//Scientific and Technological Innovation 2030/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Humans ; *Gene Knock-In Techniques ; *Lysosomal Membrane Proteins/genetics/metabolism ; *Receptors, Scavenger/genetics/metabolism ; CRISPR-Cas Systems ; Enterovirus/genetics/pathogenicity/physiology ; Hand, Foot and Mouth Disease/virology/pathology ; Coxsackievirus Infections/virology ; Neurons/virology/pathology ; Gene Editing ; },
abstract = {Hand, Foot, and Mouth Disease (HFMD) is a viral illness caused by enterovirus infections. While the introduction of the enterovirus 71 (EV71) vaccine has significantly reduced the number of EV71-related cases, the continued spread of Coxsackievirus A16 (CVA16) remains a major public health threat. Previous studies have shown that human SCARB2 (hSCARB2) knock-in (KI) mice, generated using embryonic stem cell (ESC) technology, are susceptible to CVA16. However, these models have failed to reproduce the clinical pathology and neurotoxicity after CVA16 infection. Therefore, there is an urgent need for a more reliable and effective animal model to study CVA16. In this study, we successfully created a hSCARB2 KI mouse model targeting the ROSA26 locus using CRISPR/Cas9 gene editing technology. The application of CRISPR/Cas9 enabled stable and widespread expression of hSCARB2 in the model. After infection, the KI mice exhibited a clinical pathology that closely mimics human infection, with prominent limb weakness and paralysis. The virus was detectable in multiple major organs of the mice, with peak viral load observed on day 7 post-infection, gradually clearing thereafter. Further analysis revealed widespread neuronal necrosis and infiltration of inflammatory cells in the brain and spinal cord of the KI mice. Additionally, significant activation of astrocytes (GFAP-positive) and microglia (IBA1-positive) was observed in the brain, suggesting that CVA16 infection may induce limb paralysis by attacking neuronal cells. Overall, this model effectively replicates the neuropathological changes induced by CVA16 infection and provides a potential experimental platform for studying CVA16-associated pathogenesis and neurotoxicity.},
}

Animals
Mice
*Disease Models, Animal
Humans
*Gene Knock-In Techniques
*Lysosomal Membrane Proteins/genetics/metabolism
*Receptors, Scavenger/genetics/metabolism
CRISPR-Cas Systems
Enterovirus/genetics/pathogenicity/physiology
Hand, Foot and Mouth Disease/virology/pathology
Coxsackievirus Infections/virology
Neurons/virology/pathology
Gene Editing

@article {pmid39543397,
year = {2025},
author = {Loveless, TB and Carlson, CK and Dentzel Helmy, CA and Hu, VJ and Ross, SK and Demelo, MC and Murtaza, A and Liang, G and Ficht, M and Singhai, A and Pajoh-Casco, MJ and Liu, CC},
title = {Open-ended molecular recording of sequential cellular events into DNA.},
journal = {Nature chemical biology},
volume = {21},
number = {4},
pages = {512-521},
pmid = {39543397},
issn = {1552-4469},
support = {Predoctoral Fellowship//American Heart Association (American Heart Association, Inc.)/ ; R00GM140254//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DP2 GM119163/GM/NIGMS NIH HHS/United States ; DP2GM119163//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GRFP//National Science Foundation (NSF)/ ; R35 GM136297/GM/NIGMS NIH HHS/United States ; K99GM140254//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21GM126287//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21 GM126287/GM/NIGMS NIH HHS/United States ; 1763272//National Science Foundation (NSF)/ ; },
mesh = {*DNA/genetics ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Animals ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences by DNA sequencing. We present a DNA recorder, peCHYRON, that achieves high-information, durable, and temporally resolved multiplexed recording of multiple cellular signals in mammalian cells. In each step of recording, prime editor, a Cas9-reverse transcriptase fusion protein, inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Insertions accumulate sequentially in a unidirectional order, editing can continue indefinitely, and high information is achieved by coexpressing a variety of prime editing guide RNAs (pegRNAs), each harboring unique triplet DNA sequences. We demonstrate that the constitutive expression of pegRNA collections generates insertion patterns for the straightforward reconstruction of cell lineage relationships and that the inducible expression of specific pegRNAs results in the accurate recording of exposures to biological stimuli.},
}

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

@article {pmid40143178,
year = {2025},
author = {Elshobary, ME and Badawy, NK and Ashraf, Y and Zatioun, AA and Masriya, HH and Ammar, MM and Mohamed, NA and Mourad, S and Assy, AM},
title = {Combating Antibiotic Resistance: Mechanisms, Multidrug-Resistant Pathogens, and Novel Therapeutic Approaches: An Updated Review.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {3},
pages = {},
doi = {10.3390/ph18030402},
pmid = {40143178},
issn = {1424-8247},
abstract = {The escalating global health crisis of antibiotic resistance, driven by the rapid emergence of multidrug-resistant (MDR) bacterial pathogens, necessitates urgent and innovative countermeasures. This review comprehensively examines the diverse mechanisms employed by bacteria to evade antibiotic action, including alterations in cell membrane permeability, efflux pump overexpression, biofilm formation, target site modifications, and the enzymatic degradation of antibiotics. Specific focus is given to membrane transport systems such as ATP-binding cassette (ABC) transporters, resistance-nodulation-division (RND) efflux pumps, major facilitator superfamily (MFS) transporters, multidrug and toxic compound extrusion (MATE) systems, small multidrug resistance (SMR) families, and proteobacterial antimicrobial compound efflux (PACE) families. Additionally, the review explores the global burden of MDR pathogens and evaluates emerging therapeutic strategies, including quorum quenching (QQ), probiotics, postbiotics, synbiotics, antimicrobial peptides (AMPs), stem cell applications, immunotherapy, antibacterial photodynamic therapy (aPDT), and bacteriophage. Furthermore, this review discusses novel antimicrobial agents, such as animal-venom-derived compounds and nanobiotics, as promising alternatives to conventional antibiotics. The interplay between clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) in bacterial adaptive immunity is analyzed, revealing opportunities for targeted genetic interventions. By synthesizing current advancements and emerging strategies, this review underscores the necessity of interdisciplinary collaboration among biomedical scientists, researchers, and the pharmaceutical industry to drive the development of novel antibacterial agents. Ultimately, this comprehensive analysis provides a roadmap for future research, emphasizing the urgent need for sustainable and cooperative approaches to combat antibiotic resistance and safeguard global health.},
}

@article {pmid40142492,
year = {2025},
author = {Sadanov, AK and Baimakhanova, BB and Orasymbet, SE and Ratnikova, IA and Turlybaeva, ZZ and Baimakhanova, GB and Amitova, AA and Omirbekova, AA and Aitkaliyeva, GS and Kossalbayev, BD and Belkozhayev, AM},
title = {Engineering Useful Microbial Species for Pharmaceutical Applications.},
journal = {Microorganisms},
volume = {13},
number = {3},
pages = {},
doi = {10.3390/microorganisms13030599},
pmid = {40142492},
issn = {2076-2607},
support = {BR21882248//Program-targeted funding of the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakh-stan/ ; },
abstract = {Microbial engineering has made a significant breakthrough in pharmaceutical biotechnology, greatly expanding the production of biologically active compounds, therapeutic proteins, and novel drug candidates. Recent advancements in genetic engineering, synthetic biology, and adaptive evolution have contributed to the optimization of microbial strains for pharmaceutical applications, playing a crucial role in enhancing their productivity and stability. The CRISPR-Cas system is widely utilized as a precise genome modification tool, enabling the enhancement of metabolite biosynthesis and the activation of synthetic biological pathways. Additionally, synthetic biology approaches allow for the targeted design of microorganisms with improved metabolic efficiency and therapeutic potential, thereby accelerating the development of new pharmaceutical products. The integration of artificial intelligence (AI) and machine learning (ML) plays a vital role in further advancing microbial engineering by predicting metabolic network interactions, optimizing bioprocesses, and accelerating the drug discovery process. However, challenges such as the efficient optimization of metabolic pathways, ensuring sustainable industrial-scale production, and meeting international regulatory requirements remain critical barriers in the field. Furthermore, to mitigate potential risks, it is essential to develop stringent biocontainment strategies and implement appropriate regulatory oversight. This review comprehensively examines recent innovations in microbial engineering, analyzing key technological advancements, regulatory challenges, and future development perspectives.},
}

@article {pmid40141287,
year = {2025},
author = {Li, J and Zhang, L and Li, C and Chen, W and Wang, T and Tan, L and Qiu, Y and Song, S and Li, B and Li, L},
title = {The Pentatricopeptide Repeat Protein OsPPR674 Regulates Rice Growth and Drought Sensitivity by Modulating RNA Editing of the Mitochondrial Transcript ccmC.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062646},
pmid = {40141287},
issn = {1422-0067},
support = {U24A20396//National Natural Science Foundation of China/ ; 2024CX119//Hunan agricultural science and technology innovation fund project/ ; },
mesh = {*Oryza/genetics/growth & development/metabolism ; *RNA Editing ; *Plant Proteins/genetics/metabolism ; *Droughts ; *Gene Expression Regulation, Plant ; *Mitochondria/metabolism/genetics ; RNA, Mitochondrial/metabolism/genetics ; Mitochondrial Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Mutation ; },
abstract = {The P-type pentatricopeptide repeat (PPR) proteins are crucial for RNA editing and post-transcriptional regulation in plant organelles, particularly mitochondria. This study investigates the role of OsPPR674 in rice, focusing on its function in mitochondrial RNA editing. Using CRISPR/Cas9 technology, we generated ppr674 mutant and examined its phenotypic and molecular characteristics. The results indicate that ppr674 exhibits reduced plant height, decreased seed-setting rate, and poor drought tolerance. Further analysis revealed that in the ppr674 mutant, RNA editing at the 299th nucleotide position of the mitochondrial ccmC gene (C-to-U conversion) was abolished. REMSAs showed that GST-PPR674 specifically binds to RNA probes targeting this ccmC-299 site, confirming its role in this editing process. In summary, these results suggest that OsPPR674 plays a pivotal role in mitochondrial RNA editing, emphasizing the significance of PPR proteins in organelle function and plant development.},
}

*Oryza/genetics/growth & development/metabolism
*RNA Editing
*Plant Proteins/genetics/metabolism
*Droughts
*Gene Expression Regulation, Plant
*Mitochondria/metabolism/genetics
RNA, Mitochondrial/metabolism/genetics
Mitochondrial Proteins/metabolism/genetics
CRISPR-Cas Systems
Plants, Genetically Modified
Mutation

@article {pmid40141236,
year = {2025},
author = {Livneh, Y and Agmon, D and Leor-Librach, E and Vainstein, A},
title = {Viral-Based Gene Editing System for Nutritional Improvement of Fructan Content in Lettuce.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062594},
pmid = {40141236},
issn = {1422-0067},
support = {12-01-0037//Office of the Chief Scientist/ ; 20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Fructans/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Plant Viruses/genetics ; },
abstract = {Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar 'Noga'. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The system achieved an average editing efficiency of 48.7%, with up to 78.9% of regenerated plantlets showing independent mutations. This approach eliminates the need for antibiotic selection, simplifying tissue culture processes. The system supports diverse applications, including Cas12a editing and large-fragment deletions using dual gRNA sets. Targeting the fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines with significant increases in prebiotic dietary fibre fructan content, up to 5.2-fold, and an average rise in the degree of polymerisation by 2.15 units compared with controls. Combining 1-FEH1 and 1-FEH2 knockouts did not further increase fructan levels, revealing 1-FEH2 as the predominant isozyme in lettuce. RT-qPCR analysis showed reduced expression of the upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition in fructan metabolism. This TRV-based gene editing approach, utilised here to increase fructan content, could be applied to improve other valuable traits in lettuce, and may inspire similar systems to enhance nutritional content of crops.},
}

*Lactuca/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Fructans/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Plant Viruses/genetics

@article {pmid40141178,
year = {2025},
author = {Tan, Q and Wang, J and Hao, Y and Yang, S and Cao, B and Pan, W and Cao, M},
title = {Elf1 Deficiency Impairs Macrophage Development in Zebrafish Model Organism.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062537},
pmid = {40141178},
issn = {1422-0067},
support = {2023YFA1802000//National Key R&D Program of China/ ; ZDBS-LY-SM010//Key Research Program of Frontier Sciences, Chinese Academy of Sciences/ ; YSBR-077//CAS Project for Young Scientists in Basic Research/ ; 21JC1406300//Shanghai Science and Technology In-novation Action Plan for Basic Research Program/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Macrophages/metabolism ; *Zebrafish Proteins/genetics/metabolism/deficiency ; Transcription Factors/genetics/metabolism/deficiency ; Receptors, CXCR4/genetics/metabolism ; Cell Differentiation/genetics ; Cell Movement/genetics ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockdown Techniques ; },
abstract = {The Ets (E-twenty-six) family of transcription factors plays a critical role in hematopoiesis and myeloid differentiation. However, the specific functions of many family members in these processes remain largely underexplored and poorly understood. Here, we identify Elf1 (E74-like factor 1), an Ets family member, as a critical regulator of macrophage development in the zebrafish model organism, with minimal impact on neutrophil differentiation. Through morpholino knockdown screening and CRISPR/Cas9-mediated gene editing, we demonstrate that Elf1 is critical for macrophage development and tissue injury responses. Specific overexpression of dominant-negative Elf1 (DN-Elf1) in macrophages demonstrated a cell-autonomous effect on macrophage infiltration. Furthermore, the overexpression of cxcr4b, a gene downstream of Elf1 regulation and essential for cell migration and injury response, significantly rescued this defect, indicating Elf1 as a key regulator of macrophage function. Our findings shed light on the roles of Elf1 in macrophage development and injury response and also highlight zebrafish as a powerful model for immunity research.},
}

Animals
*Zebrafish/genetics
*Macrophages/metabolism
*Zebrafish Proteins/genetics/metabolism/deficiency
Transcription Factors/genetics/metabolism/deficiency
Receptors, CXCR4/genetics/metabolism
Cell Differentiation/genetics
Cell Movement/genetics
CRISPR-Cas Systems
Gene Editing
Gene Knockdown Techniques

@article {pmid40141150,
year = {2025},
author = {Rendueles, C and Garay-Novillo, JN and Rau, MH and Gaspar, P and Ruiz-Masó, JÁ and Mahony, J and Rodríguez, A and Barra, JL and Del Solar, G and Martínez, B},
title = {A Plasmid-Encoded Surface Polysaccharide Partly Blocks Ceduovirus Infection in Lactococci.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062508},
pmid = {40141150},
issn = {1422-0067},
support = {PID2020-119697RB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; },
mesh = {*Lactococcus/genetics ; *Plasmids/genetics ; *Lactococcus lactis/genetics/virology ; Bacteriophages/genetics ; Polysaccharides, Bacterial/metabolism ; CRISPR-Cas Systems ; },
abstract = {Bacteriophages (or phages) remain the leading cause of failure in dairy fermentations. Thereby, phage-resistant Lactococcus lactis and Lactococcus cremoris dairy starters are in continuous demand. In this work, our goal was to identify phage defense mechanisms against ceduoviruses encoded by two wild isolates of dairy origin named L. lactis IPLA517 and IPLA1064. These strains were previously subjected to experimental evolution to select derivatives that are resistant to the bacteriocin Lcn972. It was observed that the Lcn972[R] derivatives became sensitive to phage infection; however, the underlying mechanism was not defined. The long-read sequencing technologies applied in this work reveal that all of the Lcn972[R] derivatives shared the loss of a 41 kb endogenous plasmid (p41) that harbors a putative exopolysaccharide (EPS) gene cluster with significant homology to one described in Lactococcus garvieae. Using a CRISPR-Cas9-based approach, p41 was selectively cured from L. lactis IPLA1064. Phage infection assays with three ceduoviruses demonstrated that curing p41 restored phage sensitivity at levels comparable to the Lcn972[R]-IPLA1064 derivatives. Phage adsorption to Δp41 cells was also increased, consistent with the hypothesis of EPS production hindering access to the phage receptor protein Pip. Our results reinforce the role of EPSs in protecting Lactococcus against phage infection, a phenomenon that is rarely reported for ceduoviruses. Moreover, the results also exemplify the likely horizontal gene transfer that can occur between L. lactis and L. garvieae in a dairy environment.},
}

*Lactococcus/genetics
*Plasmids/genetics
*Lactococcus lactis/genetics/virology
Bacteriophages/genetics
Polysaccharides, Bacterial/metabolism
CRISPR-Cas Systems

@article {pmid40141141,
year = {2025},
author = {Lin, K and Lu, LX and Pan, BZ and Chai, X and Fu, QT and Geng, XC and Mo, Y and Fei, YC and Xu, JJ and Li, M and Ni, J and Xu, ZF},
title = {Agrobacterium rhizogenes-Mediated Hairy Root Genetic Transformation Using Agrobacterium Gel Inoculation and RUBY Reporter Enables Efficient Gene Function Analysis in Sacha Inchi (Plukenetia volubilis).},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062496},
pmid = {40141141},
issn = {1422-0067},
support = {AD23026337//Guangxi Specific Project for Science and Technology Bases and Talents/ ; XDA24030502//Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding)/ ; YNWR-QNBJ-2020-172//West Light Foundation of the Chinese Academy of Sciences, the Youth Talent Support Program of Yunnana Province/ ; },
mesh = {*Agrobacterium/genetics ; *Plant Roots/genetics/microbiology/metabolism ; *Transformation, Genetic ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Fusarium/genetics ; Plant Proteins/genetics/metabolism ; Genes, Reporter ; Gene Editing/methods ; },
abstract = {Plukenetia volubilis L., a woody oilseed plant rich in α-linolenic acid, represents a promising source of polyunsaturated fatty acids. However, the lack of an efficient genetic transformation system has significantly hindered gene function research and molecular breeding in P. volubilis. In this study, we developed a highly efficient Agrobacterium rhizogenes-mediated hairy root transformation system for P. volubilis via the use of Agrobacterium gel in combination with the visually detectable RUBY reporter for gene function analysis in roots. The results indicate that the optimal transformation method involves infecting P. volubilis seedlings with Agrobacterium gel containing acetosyringone and inducing hairy root formation in perlite. This approach resulted in more than 18.97% of the seedlings producing positive hairy roots overexpressing the RUBY gene. Using this genetic transformation system, we successfully overexpressed the antimicrobial peptide-encoding gene CEMA in hairy roots, which enhanced the resistance of P. volubilis to Fusarium oxysporum. Furthermore, by combining this transformation system with the CRISPR-Cas9 tool, we validated the regulatory role of PvoSHR in the development of root epidermal cells in P. volubilis. Unexpectedly, a 123-bp DNA fragment from the T-DNA region of the A. rhizogenes Ri plasmid was found to be knocked in to the P. volubilis genome, replacing a 110-bp fragment of PvoSHR at CRISPR-Cas9 induced double-strand DNA breaks. Conclusively, this system provides a powerful tool for gene function research in P. volubilis and provides novel insights into the development of transformation and gene editing systems for other woody plants.},
}

*Agrobacterium/genetics
*Plant Roots/genetics/microbiology/metabolism
*Transformation, Genetic
*Plants, Genetically Modified/genetics
CRISPR-Cas Systems
Fusarium/genetics
Plant Proteins/genetics/metabolism
Genes, Reporter
Gene Editing/methods

@article {pmid40140116,
year = {2025},
author = {Anyaegbunam, NJ and Okpe, KE and Bello, AB and Ajanaobionye, TI and Mgboji, CC and Olonade, A and Anyaegbunam, ZKG and Mba, IE},
title = {Leveraging innovative diagnostics as a tool to contain superbugs.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {4},
pages = {63},
pmid = {40140116},
issn = {1572-9699},
mesh = {Humans ; *Bacteria/genetics/isolation & purification ; Bacterial Infections/diagnosis/microbiology ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The evolutionary adaptation of pathogens to biological materials has led to an upsurge in drug-resistant superbugs that significantly threaten public health. Treating most infections is an uphill task, especially those associated with multi-drug-resistant pathogens, biofilm formation, persister cells, and pathogens that have acquired robust colonization and immune evasion mechanisms. Innovative diagnostic solutions are crucial for identifying and understanding these pathogens, initiating efficient treatment regimens, and curtailing their spread. While next-generation sequencing has proven invaluable in diagnosis over the years, the most glaring drawbacks must be addressed quickly. Many promising pathogen-associated and host biomarkers hold promise, but their sensitivity and specificity remain questionable. The integration of CRISPR-Cas9 enrichment with nanopore sequencing shows promise in rapid bacterial diagnosis from blood samples. Moreover, machine learning and artificial intelligence are proving indispensable in diagnosing pathogens. However, despite renewed efforts from all quarters to improve diagnosis, accelerated bacterial diagnosis, especially in Africa, remains a mystery to this day. In this review, we discuss current and emerging diagnostic approaches, pinpointing the limitations and challenges associated with each technique and their potential to help address drug-resistant bacterial threats. We further critically delve into the need for accelerated diagnosis in low- and middle-income countries, which harbor more infectious disease threats. Overall, this review provides an up-to-date overview of the diagnostic approaches needed for a prompt response to imminent or possible bacterial infectious disease outbreaks.},
}

Humans
*Bacteria/genetics/isolation & purification
Bacterial Infections/diagnosis/microbiology
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems
Drug Resistance, Multiple, Bacterial/genetics
Anti-Bacterial Agents/pharmacology

@article {pmid40138716,
year = {2025},
author = {Whitford, CM and Gockel, P and Faurdal, D and Gren, T and Sigrist, R and Weber, T},
title = {CASCADE-Cas3 enables highly efficient genome engineering in Streptomyces species.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf214},
pmid = {40138716},
issn = {1362-4962},
support = {NNF20CC0035580//Novo Nordisk Foundation/ ; //DTU Biosustain/ ; },
mesh = {*Streptomyces/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Bacterial/genetics ; *Plasmids/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Engineering/methods ; },
abstract = {Type I clustered regularly interspaced short palindromic repeat (CRISPR) systems are widespread in bacteria and archaea. Compared to more widely applied type II systems, type I systems differ in the multi-effector CRISPR-associated complex for antiviral defense needed for crRNA processing and target recognition, as well as the processive nature of the hallmark nuclease Cas3. Given the widespread nature of type I systems, the processive nature of Cas3 and the recombinogenic overhangs created by Cas3, we hypothesized that CASCADE-Cas3 would be uniquely positioned to enable efficient genome engineering in streptomycetes. Here, we report a new type I based CRISPR genome engineering tool for streptomycetes. The plasmid system, called pCRISPR-Cas3, utilizes a compact type I-C CRISPR system and enables highly efficient genome engineering. pCRISPR-Cas3 outperforms pCRISPR-Cas9 and facilitates targeted and random sized deletions. Furthermore, we demonstrate its ability to effectively perform substitutions of large genomic regions such as biosynthetic gene clusters. Without additional modifications, pCRISPR-Cas3 enabled genome engineering in several Streptomyces species at high efficiencies.},
}

*Streptomyces/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Genome, Bacterial/genetics
*Plasmids/genetics
CRISPR-Associated Proteins/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genetic Engineering/methods

@article {pmid40138431,
year = {2025},
author = {Fuchs, W and Assad-Garcia, N and Abkallo, HM and Xue, Y and Oldfield, LM and Fedorova, N and Hübner, A and Kabuuka, T and Pannhorst, K and Höper, D and Nene, V and Gonzalez-Juarbe, N and Steinaa, L and Vashee, S},
title = {A synthetic genomics-based African swine fever virus engineering platform.},
journal = {Science advances},
volume = {11},
number = {13},
pages = {eadu7670},
doi = {10.1126/sciadv.adu7670},
pmid = {40138431},
issn = {2375-2548},
mesh = {*African Swine Fever Virus/genetics ; Animals ; Swine ; *Genomics/methods ; *African Swine Fever/virology/prevention & control ; *Genome, Viral ; *CRISPR-Cas Systems ; Reverse Genetics/methods ; Genetic Engineering/methods ; Viral Vaccines/genetics/immunology ; Gene Editing/methods ; },
abstract = {African swine fever (ASF) is a deadly viral disease in domestic pigs that has a large global economic impact for the swine industry. It is present in Africa, Europe, Asia, and in the Caribbean island of Hispaniola. There are no effective treatments or broadly licensed vaccines to prevent disease. Efforts to counteract ASF have been hampered because of the lack of convenient tools to engineer its etiological agent, ASF virus (ASFV), largely due to its large noninfectious genome. Here, we report the use of synthetic genomics methodology to develop a reverse genetics system for ASFV using a CRISPR-Cas9-inhibited self-helper virus to reconstitute live recombinant ASFV from synthetic genomes to rapidly generate a variety of combinatorial mutants of ASFV. The method will substantially facilitate the development of therapeutics or subunit and live-attenuated vaccines for ASF. This synthetic genomics-based approach has wide-ranging impact because it can be applied to rapidly develop reverse genetics tools for emerging viruses with noninfectious genomes.},
}

*African Swine Fever Virus/genetics
Animals
Swine
*Genomics/methods
*African Swine Fever/virology/prevention & control
*Genome, Viral
*CRISPR-Cas Systems
Reverse Genetics/methods
Genetic Engineering/methods
Viral Vaccines/genetics/immunology
Gene Editing/methods

@article {pmid40138429,
year = {2025},
author = {Iacovacci, J and Brough, R and Moughari, FA and Alexander, J and Kemp, H and Tutt, ANJ and Natrajan, R and Lord, CJ and Haider, S},
title = {Proteogenomic discovery of RB1-defective phenocopy in cancer predicts disease outcome, response to treatment, and therapeutic targets.},
journal = {Science advances},
volume = {11},
number = {13},
pages = {eadq9495},
doi = {10.1126/sciadv.adq9495},
pmid = {40138429},
issn = {2375-2548},
mesh = {Humans ; *Proteogenomics/methods ; *Retinoblastoma Binding Proteins/genetics/metabolism ; *Neoplasms/genetics/drug therapy/metabolism ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Cell Line, Tumor ; Retinoblastoma Protein/genetics/metabolism ; Treatment Outcome ; Gene Expression Regulation, Neoplastic ; Phenotype ; CRISPR-Cas Systems ; Molecular Targeted Therapy ; Mutation ; Prognosis ; Female ; },
abstract = {Genomic defects caused by truncating mutations or deletions in the Retinoblastoma tumor suppressor gene (RB1) are frequently observed in many cancer types leading to dysregulation of the RB pathway. Here, we propose an integrative proteogenomic approach that predicts cancers with dysregulation in the RB pathway. A subset of these cancers, which we term as "RBness," lack RB1 genomic defects and yet phenocopy the transcriptional profile of RB1-defective cancers. We report RBness as a pan-cancer phenomenon, associated with patient outcome and chemotherapy response in multiple cancer types, and predictive of CDK4/6 inhibitor response in estrogen-positive breast cancer. Using RNA interference and a CRISPR-Cas9 screen in isogenic models, we find that RBness cancers also phenocopy synthetic lethal vulnerabilities of cells with RB1 genomic defects. In summary, our findings suggest that dysregulation of the RB pathway in cancers lacking RB1 genomic defects provides a molecular rationale for how these cancers could be treated.},
}

Humans
*Proteogenomics/methods
*Retinoblastoma Binding Proteins/genetics/metabolism
*Neoplasms/genetics/drug therapy/metabolism
*Ubiquitin-Protein Ligases/genetics/metabolism
Cell Line, Tumor
Retinoblastoma Protein/genetics/metabolism
Treatment Outcome
Gene Expression Regulation, Neoplastic
Phenotype
CRISPR-Cas Systems
Molecular Targeted Therapy
Mutation
Prognosis
Female

@article {pmid40138428,
year = {2025},
author = {Tian, R and Tian, X and Yang, M and Song, Y and Zhao, T and Zhong, C and Zhu, W and Zhou, P and Han, Z and Hu, Z},
title = {Systematic high-throughput evaluation reveals FrCas9's superior specificity and efficiency for therapeutic genome editing.},
journal = {Science advances},
volume = {11},
number = {13},
pages = {eadu7334},
doi = {10.1126/sciadv.adu7334},
pmid = {40138428},
issn = {2375-2548},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Genetic Therapy/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing ; Animals ; },
abstract = {CRISPR-Cas9 systems have revolutionized genome editing, but the off-target effects of Cas9 limit its use in clinical applications. Here, we systematically evaluate FrCas9, a variant from Faecalibaculum rodentium, for cell and gene therapy (CGT) applications and compare its performance to SpCas9 and OpenCRISPR-1. OpenCRISPR-1 is a CRISPR system synthesized de novo using large language models (LLMs) but has not yet undergone systematic characterization. Using AID-seq, Amplicon sequencing, and GUIDE-seq, we assessed the on-target activity and off-target profiles of these systems across multiple genomic loci. FrCas9 demonstrated higher on-target efficiency and substantially fewer off-target effects than SpCas9 and OpenCRISPR-1. Furthermore, TREX2 fusion with FrCas9 reduced large deletions and translocations, enhancing genomic stability. Through screening of 1903 sgRNAs targeting 21 CGT-relevant genes using sequential AID-seq, Amplicon sequencing, and GUIDE-seq analysis, we identified optimal sgRNAs for each gene. Our high-throughput screening platform highlights FrCas9, particularly in its TREX2-fused form, as a highly specific and efficient tool for precise therapeutic genome editing.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
Genetic Therapy/methods
CRISPR-Associated Protein 9/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing
Animals

@article {pmid40138366,
year = {2025},
author = {Rivera-Toro, DM and de Folter, S and Alvarez-Venegas, R},
title = {CRISPR/dCas12a-mediated activation of SlPAL2 enhances tomato resistance against bacterial canker disease.},
journal = {PloS one},
volume = {20},
number = {3},
pages = {e0320436},
doi = {10.1371/journal.pone.0320436},
pmid = {40138366},
issn = {1932-6203},
mesh = {*Solanum lycopersicum/microbiology/genetics/immunology ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plants, Genetically Modified ; *Phenylalanine Ammonia-Lyase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Clavibacter/genetics ; Gene Expression Regulation, Plant ; Lignin/metabolism ; },
abstract = {Crop protection is essential for maintaining and improving agricultural productivity. While pesticides are commonly used to control pests, they pose several challenges, including environmental harm and health risks. Alternative strategies to pesticides include breeding resistant crop varieties, biological control, and utilizing genome-editing tools like CRISPR/Cas. However, the application of epigenome editing, particularly CRISPR activation (CRISPRa), in plants remains underexplored. Phenylalanine ammonia-lyase (PAL), a key enzyme in the phenylpropanoid pathway, plays a pivotal role in plant defense by producing lignin and other secondary metabolites essential for pathogen resistance. In this study, we engineered tomato plants by fusing the SET-domain of the SlATX1 coding gene, a histone H3 lysine 4 tri-methyltransferase, to dCas12a, targeting the SlPAL2 promoter with the aim to increase PAL2 gene expression. CRISPRa-edited plants demonstrated increased deposition of the H3K4me3 epigenetic mark and significantly upregulated SlPAL2 expression. This enhanced lignin accumulation and conferred increased resistance to Clavibacter michiganensis subsp. michiganensis (Cmm) without significant reduction in plant height or fruit yield. Disease resistance was also associated with reduced pathogen load and lesion size, and higher lignin levels persisted even after SlPAL2 expression declined post-infection. These findings highlight the potential of CRISPRa for reprogramming plant defense responses through targeted histone modifications, offering a sustainable approach for crop improvement. Furthermore, CRISPRa could also be applied to enhance crop resilience in other contexts, such as addressing food security challenges by enhancing productivity.},
}

*Solanum lycopersicum/microbiology/genetics/immunology
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Plants, Genetically Modified
*Phenylalanine Ammonia-Lyase/genetics/metabolism
Plant Proteins/genetics/metabolism
Clavibacter/genetics
Gene Expression Regulation, Plant
Lignin/metabolism

@article {pmid40136952,
year = {2025},
author = {Xin, X and Su, J and Cui, H and Wang, L and Song, S},
title = {Recent Advances in Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated Proteins System-Based Biosensors.},
journal = {Biosensors},
volume = {15},
number = {3},
pages = {},
pmid = {40136952},
issn = {2079-6374},
support = {2023YFB3208200//National Key Research and Development Program of China/ ; 21974147//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
abstract = {High-sensitivity and high-specificity biodetection is critical for advancing applications in life sciences, biosafety, food safety, and environmental monitoring. CRISPR/Cas systems have emerged as transformative tools in biosensing due to their unparalleled specificity, programmability, and unique enzymatic activities. They exhibit two key cleavage behaviors: precise ON-target cleavage guided by specific protospacers, which ensures accurate target recognition, and bystander cleavage activity triggered upon target binding, which enables robust signal amplification. These properties make CRISPR/Cas systems highly versatile for designing biosensors for ultra-sensitive detection. This review comprehensively explores recent advancements in CRISPR/Cas system-based biosensors, highlighting their impact on improving biosensing performance. We discuss the integration of CRISPR/Cas systems with diverse signal readout mechanisms, including electrochemical, fluorescent, colorimetric, surface-enhanced Raman scattering (SERS), and so on. Additionally, we examine the development of integrated biosensing systems, such as microfluidic devices and portable biosensors, which leverage CRISPR/Cas technology for point-of-care testing (POCT) and high-throughput analysis. Furthermore, we identify unresolved challenges, aiming to inspire innovative solutions and accelerate the translation of these technologies into practical applications for diagnostics, food, and environment safety.},
}

*Biosensing Techniques
*CRISPR-Cas Systems
CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Humans

@article {pmid40135905,
year = {2025},
author = {Zhang, J-Z and Li, Y-Z and Xi, Z-N and Zhang, Y and Liu, Z-Y and Ma, X-Q and Li, F-L},
title = {Inducible promoters of bacterial microcompartments improve the CRISPR/Cas9 tools for efficient metabolic engineering of Clostridium ljungdahlii.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0218324},
doi = {10.1128/aem.02183-24},
pmid = {40135905},
issn = {1098-5336},
abstract = {UNLABELLED: Clostridium ljungdahlii, as a model acetogen strain, represents a novel platform for biotechnological production for CO2 fixation. The genome of C. ljungdahlii harbors two gene loci associated with glycyl radical enzyme-associated microcompartments (GRMs), which are predicted to play essential roles in choline and 1,2-propanediol (1,2-PD) metabolism. This study validated the functions of these GRM loci and identified two inducible promoters, of which Pcholine1 was induced by choline, while P1,2-PD was induced by 1,2-PD. Subsequently, the highly expressed P1,2-PD and tightly controlled Pcholine1 were applied to improve CRISPR/Cas9 gene editing tools. Specifically, P1,2-PD was used to develop a highly efficient gene knockout tool based on an all-in-one plasmid, achieving 100% deletion efficiency for multiple genes, including pyrE, pduS, aor2, and eutT. On the other hand, the cas9 gene was integrated downstream of Pcholine1 into the genome. The integrated cas9 efficiently mediated gene editing in C. ljungdahlii by introducing plasmids containing a gRNA cassette along with the relevant homology arms. This was exemplified by the construction of the Δbdh::pdc strain, where the 2,3-butanediol dehydrogenase gene was replaced with a pyruvate decarboxylase gene from Zymomonas mobilis and the 3-HB Syn KI strain, in which an artificial 3-hydroxybutyric acid synthesis pathway was inserted into the genome. This study highlights the effectiveness and convenience of the inducible CRISPR/Cas9 gene editing systems, thereby enriching the CRISPR/Cas toolkit in acetogens.

IMPORTANCE: A CRISPR/Cas9 genetic tool controlled by a constitutive promoter has been developed for precise gene deletion in Clostridium ljungdahlii. However, its efficiency was hindered by the toxicity resulting from the constitutive expression of cas9 and the large plasmids, leading to a low overall success rate. Inducible promoters, which allow for the transcription of target genes to be switched on and off in the presence or absence of inducers, have a broad range of applications. In this study, we identify two inducible promoters and apply them to enhance the CRISPR/Cas9 tools. The improved CRISPR/Cas9 tools facilitate gene editing with high efficiency, potentially playing significant roles in advancing genetic research and metabolic engineering of C. ljungdahlii.},
}

@article {pmid40135490,
year = {2025},
author = {Gou, S and Liu, Y and Li, Q and Qiu, L and Liu, Z and Zhao, Y},
title = {CRISPR/Cas12-mediated detection of GI and GII Norovirus in different food samples.},
journal = {Journal of food science},
volume = {90},
number = {3},
pages = {e70160},
doi = {10.1111/1750-3841.70160},
pmid = {40135490},
issn = {1750-3841},
support = {22N31900200//Shanghai Science and Technology Innovation action plan in the agricultural field of 2022/ ; },
mesh = {*Norovirus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Sensitivity and Specificity ; Humans ; Food Microbiology/methods ; Molecular Diagnostic Techniques ; },
abstract = {Norovirus is one of the leading causes of infectious diarrhea, occurring in about 18% of diarrhea cases worldwide. Norovirus is characterized by a low infectious dose, rapid onset, and strong transmission capacity. Given the lack of specific drugs and vaccines, developing efficient and accurate detection technologies is of great significance to prevent and control the spread of diseases. This study combined the reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology with the clustered regularly interspaced short palindromic repeats (CRISPR) technology to develop a sensitive and rapid detection method, which can reduce the reliance on temperature control and expensive real-time fluorescent polymerase chain reaction (PCR) devices. The RT-LAMP/CRISPR Cas12a method demonstrated good specificity and sensitivity, testing food samples of three different substrates with 100% positive qualitative accuracy. The detection sensitivity is 32.8 copies/reaction for Norovirus GI and 22.8 copies/reaction for Norovirus GII. This method helps to effectively identify food products contaminated with Norovirus, thereby preventing human infections and economic losses due to disease outbreaks.},
}

*Norovirus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Food Contamination/analysis
Sensitivity and Specificity
Humans
Food Microbiology/methods
Molecular Diagnostic Techniques

@article {pmid40134118,
year = {2025},
author = {Yang, F and Wang, Z and Li, D and Gao, F and Hou, X},
title = {Otud6a Knockout Leads to Male Subfertility in Mice.},
journal = {Molecular reproduction and development},
volume = {92},
number = {3},
pages = {e70022},
doi = {10.1002/mrd.70022},
pmid = {40134118},
issn = {1098-2795},
support = {//This research was supported by the National Key R&D Program of China (Grant 2021YFC2700200)./ ; },
mesh = {Animals ; Male ; *Mice, Knockout ; Mice ; *Infertility, Male/genetics/pathology/metabolism ; *Spermatogenesis/genetics ; *Sperm Motility/genetics ; Apoptosis ; Testis/metabolism/pathology ; Sperm Count ; CRISPR-Cas Systems ; Deubiquitinating Enzymes/metabolism/genetics ; },
abstract = {Ovarian tumor ubiquitinating 6A (OTUD6A) is a deubiquitinating enzyme whose aberrant expression has been linked to various diseases, including inflammation and prostate cancer. Research indicates that deubiquitinating enzymes (DUBs) play a significant role in spermatogenesis in mice. However, the role of OTUD6A in spermatogenesis remains unclear. To investigate the function of OTUD6A in mouse spermatogenesis, we generated Otud6a-knockout mice using the CRISPR/Cas9 system. Our results showed that OTUD6A is predominantly expressed in the testis and localized to the cytoplasm of spermatogonia and spermatocytes. Although no significant differences were observed in testicular size or morphology between Otud6a-knockout and wild-type mice, the knockout mice exhibited increased germ cell apoptosis, decreased epididymal sperm counts, abnormalities in sperm motility and subfertility. These findings indicate that Otud6a-knockout leads to male subfertility in mice.},
}

Animals
Male
*Mice, Knockout
Mice
*Infertility, Male/genetics/pathology/metabolism
*Spermatogenesis/genetics
*Sperm Motility/genetics
Apoptosis
Testis/metabolism/pathology
Sperm Count
CRISPR-Cas Systems
Deubiquitinating Enzymes/metabolism/genetics

@article {pmid40133358,
year = {2025},
author = {Chen, P and Lin, L and Lin, X and Liao, K and Qiang, J and Wang, Z and Wu, J and Li, Y and Yang, L and Yao, N and Song, H and Hong, Y and Liu, WH and Zhang, Y and Chang, X and Du, D and Xiao, C},
title = {A Csde1-Strap complex regulates plasma cell differentiation by coupling mRNA translation and decay.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2906},
pmid = {40133358},
issn = {2041-1723},
mesh = {*Cell Differentiation ; *Basic-Leucine Zipper Transcription Factors/metabolism/genetics ; Animals ; *Plasma Cells/metabolism/cytology ; *RNA Stability ; Mice ; *RNA-Binding Proteins/metabolism/genetics ; *RNA, Messenger/metabolism/genetics ; Humans ; Protein Biosynthesis ; Gene Expression Regulation ; Mice, Inbred C57BL ; B-Lymphocytes/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Upon encountering antigens, B cells may undergo multiple differentiation paths, including becoming plasma cells and memory B cells. Although it is well-known that transcription factors govern gene expression programs underpinning these fate decisions in transcriptional level, the role of post-transcriptional regulators, with a focus on RNA-binding proteins, in the fate determination are lesser known. Here we find by RNA interactome capture-coupled CRISPR/Cas9 functional screening that the Csde1-Strap complex plays an important role in plasma cell differentiation. Mechanistically, the Csde1-Strap complex establishes the expression kinetics of Bach2, a key regulator of plasma cell differentiation. Bach2 expression is rapidly induced to promote B cell expansion and then decreased to initiate plasma cell differentiation. The Csde1-Strap interaction is critical for their binding to Bach2 mRNA to couple its decay with translation to restrain the magnitude and duration of Bach2 protein expression. In the absence of Csde1 or Strap, Bach2 translation is de-coupled from mRNA decay, leading to elevated and prolonged expression of Bach2 protein and impaired plasma cell differentiation. This study thus establishes the functional RBP landscape in B cells and illustrates the fundamental importance of controlling protein expression kinetics in cell fate determination.},
}

*Cell Differentiation
*Basic-Leucine Zipper Transcription Factors/metabolism/genetics
Animals
*Plasma Cells/metabolism/cytology
*RNA Stability
Mice
*RNA-Binding Proteins/metabolism/genetics
*RNA, Messenger/metabolism/genetics
Humans
Protein Biosynthesis
Gene Expression Regulation
Mice, Inbred C57BL
B-Lymphocytes/metabolism/cytology
CRISPR-Cas Systems

@article {pmid40133266,
year = {2025},
author = {Aris, KDP and Cofsky, JC and Shi, H and Al-Sayyad, N and Ivanov, IE and Balaji, A and Doudna, JA and Bryant, Z},
title = {Dynamic basis of supercoiling-dependent DNA interrogation by Cas12a via R-loop intermediates.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2939},
pmid = {40133266},
issn = {2041-1723},
support = {R01GM106159//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; Kinetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *R-Loop Structures ; *DNA/chemistry/metabolism ; *DNA, Superhelical/chemistry/metabolism ; Endodeoxyribonucleases/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/chemistry/genetics ; Nucleic Acid Conformation ; Base Pairing ; },
abstract = {The sequence specificity and programmability of DNA binding and cleavage have enabled widespread applications of CRISPR-Cas12a in genetic engineering. As an RNA-guided CRISPR endonuclease, Cas12a engages a 20-base pair (bp) DNA segment by forming a three-stranded R-loop structure in which the guide RNA hybridizes to the DNA target. Here we use single-molecule torque spectroscopy to investigate the dynamics and mechanics of R-loop formation of two widely used Cas12a orthologs at base-pair resolution. We directly observe kinetic intermediates corresponding to a ~5 bp initial RNA-DNA hybridization and a ~17 bp intermediate preceding R-loop completion, followed by transient DNA unwinding that extends beyond the 20 bp R-loop. The complex multistate landscape of R-loop formation is ortholog-dependent and shaped by target sequence, mismatches, and DNA supercoiling. A four-state kinetic model captures essential features of Cas12a R-loop dynamics and provides a biophysical framework for understanding Cas12a activity and specificity.},
}

*CRISPR-Associated Proteins/metabolism/chemistry
Kinetics
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/metabolism
*R-Loop Structures
*DNA/chemistry/metabolism
*DNA, Superhelical/chemistry/metabolism
Endodeoxyribonucleases/metabolism/chemistry/genetics
Bacterial Proteins/metabolism/chemistry/genetics
Nucleic Acid Conformation
Base Pairing

@article {pmid40133194,
year = {2025},
author = {Wang, KC and Zheng, T and Hubbard, BP},
title = {CRISPR/Cas technologies for cancer drug discovery and treatment.},
journal = {Trends in pharmacological sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tips.2025.02.009},
pmid = {40133194},
issn = {1873-3735},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) tools are revolutionizing the establishment of genotype-phenotype relationships and are transforming cell- and gene-based therapies. In the field of oncology, CRISPR/CRISPR-associated protein 9 (Cas9), Cas12, and Cas13 have advanced the generation of cancer models, the study of tumor evolution, the identification of target genes involved in cancer growth, and the discovery of genes involved in chemosensitivity and resistance. Moreover, preclinical therapeutic strategies employing CRISPR/Cas have emerged. These include the generation of chimeric antigen receptor T (CAR-T) cells and engineered immune cells, and the use of precision anticancer gene-editing agents to inactivate driver oncogenes, suppress tumor support genes, and cull cancer cells in response to genetic circuit output. This review summarizes the collective impact that CRISPR technology has had on basic and applied cancer research, and highlights the promises and challenges facing its clinical translation.},
}

@article {pmid40132699,
year = {2025},
author = {Chena, F and Chenb, L},
title = {CRISPR/Cas-mediated macromolecular DNA methylation editing: Precision targeting of DNA methyltransferases in cancer therapy.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {142401},
doi = {10.1016/j.ijbiomac.2025.142401},
pmid = {40132699},
issn = {1879-0003},
abstract = {Epigenetic modifications, particularly DNA methylation, play a pivotal role in gene regulation, influencing tumor suppressor silencing and oncogene activation in cancer. DNA methyltransferases (DNMTs), Ten-eleven translocation (TET) enzymes, and associated chromatin regulators are key biological macromolecules that mediate these epigenetic processes. Targeting aberrant DNA methylation holds great promise for cancer therapy, but traditional approaches lack precision and specificity. CRISPR/Cas-based epigenetic editing has emerged as a transformative tool for macromolecular DNA methylation reprogramming, offering targeted modifications without altering the genetic sequence. This review explores the role of DNMTs, TET enzymes, and chromatin-associated proteins in cancer epigenetics and discusses how CRISPR/dCas9 fused with DNMT3A or TET1 enables locus-specific DNA methylation editing. We highlight recent advances, including dCas9-DNMT3A for precise hypermethylation and dCas9-TET1 for targeted demethylation, and discuss their applications in reactivating tumor suppressor genes or silencing oncogenic pathways. Novel epigenetic editing systems, such as SunTag-based amplification, KRAB-MeCP2 repression, further enhance targeting efficiency and therapeutic potential. CRISPR/Cas-mediated macromolecular epigenetic editing represents a paradigm shift in cancer therapy, providing unprecedented control over DNA methylation and chromatin regulation. Despite challenges such as tumor heterogeneity and off-target effects, integrating CRISPR-based methylation reprogramming with precision oncology holds immense promise for future clinical applications.},
}

@article {pmid40132543,
year = {2025},
author = {Keeley, S and Fernández-Lajarín, M and Bergemann, D and John, N and Parrott, L and Andrea, BE and González-Rosa, JM},
title = {Rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system.},
journal = {Cell reports methods},
volume = {5},
number = {3},
pages = {101003},
doi = {10.1016/j.crmeth.2025.101003},
pmid = {40132543},
issn = {2667-2375},
mesh = {Animals ; *Zebrafish/genetics ; *Myocytes, Cardiac/metabolism ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Zebrafish Proteins/genetics/metabolism ; Mutation ; Animals, Genetically Modified ; Gene Targeting/methods ; },
abstract = {CRISPR-Cas9 has accelerated loss-of-function studies in zebrafish, but creating tissue-specific mutant lines is still labor intensive. While some tissue-specific Cas9 zebrafish lines exist, standardized methods for gene targeting, including guide RNA (gRNA) delivery, are lacking, limiting broader use in the community. To tackle these limitations, we develop a cardiomyocyte-specific Cas9 line, the cardiodeleter, that efficiently generates biallelic mutations in combination with gene-specific gRNAs. We create transposon-based guide shuttles that deliver gRNAs targeting a gene of interest while permanently labeling cells susceptible to becoming mutant. We validate this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), resulting in the loss of the corresponding protein or phenocopy of established mutants. We provide detailed protocols for generating guide shuttles, facilitating the adoption of these techniques in the zebrafish community. Our approach enables rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, making it a valuable tool for cell-autonomous studies and genetic screening.},
}

Animals
*Zebrafish/genetics
*Myocytes, Cardiac/metabolism
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
Zebrafish Proteins/genetics/metabolism
Mutation
Animals, Genetically Modified
Gene Targeting/methods

@article {pmid40131645,
year = {2025},
author = {Wang, W and He, H and Liu, H and Gao, Y and Dang, F and Zhao, X and Chen, S and Li, L and Lu, Y},
title = {Developing a robust genome editing tool based on an endogenous type I-B CRISPR-Cas system in Saccharopolyspora spinosa.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {40131645},
issn = {1869-1889},
abstract = {Saccharopolyspora spinosa is an industrial rare actinomycete capable of producing important environmental-friendly biopesticides, spinosyns. However, exploitation of S. spinosa has been limited due to its genetic inaccessibility and lack of effective genome engineering tools. In this work, we characterized the activity of an endogenous type I-B CRISPR-Cas system as well as its recognized protospacer adjacent motifs (PAMs) based on bioinformatics analysis combined with a plasmid interference assay in S. spinosa. By delivering editing plasmids containing a designed miniCRISPR array (repeat+self-targeting spacer+repeat) and repair templates, we achieved 100% editing efficiency for gene deletion. Using this tool, the genetic barrier composed of the restriction-modification (RM) systems was systematically disarmed. We showed that by disarming one type I RM system (encoded by A8926_1903/1904/1905) and two type II RM systems (encoded by A8926_1725/1726 and A8926_2652/2653) simultaneously, the transformation efficiency of the replicative and integrative plasmids (pSP01 and pSI01) was increased by approximately 3.9-fold and 4.2-fold, respectively. Using the engineered strain with simultaneous knock-out of these three RM genes as the starting strain, we achieved the deletion of 75-kb spinosyns biosynthetic gene cluster (BGC) as well as gene insertion at high efficiency. Collectively, we developed a reliable and highly efficient genome editing tool based on the endogenous type I CRISPR-Cas system combined with the disarmament of the RM systems in S. spinosa. This is the first time to establish an endogenous CRISPR-Cas-based genome editing tool in the non-model industrial actinomycetes.},
}

@article {pmid40131558,
year = {2025},
author = {Yamanaka, T and Sogo, A and Maegawa, S and Kinoshita, M},
title = {Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {15},
pmid = {40131558},
issn = {1573-9368},
support = {JPJ008723//Ministry of Agriculture, Forestry and Fisheries/ ; },
mesh = {Animals ; *Oryzias/genetics/embryology ; *Zebrafish/genetics/embryology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Mutagenesis ; Embryo, Nonmammalian ; Cold Temperature ; Temperature ; Mutation ; },
abstract = {Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.},
}

Animals
*Oryzias/genetics/embryology
*Zebrafish/genetics/embryology
*Gene Editing/methods
*CRISPR-Cas Systems
*Mutagenesis
Embryo, Nonmammalian
Cold Temperature
Temperature
Mutation

@article {pmid40131505,
year = {2025},
author = {Darzi, F and Khamesipour, A and Tasbihi, M and Bahraminasab, M and Nateghi-Rostami, M},
title = {CRISPR/Cas9-mediated deletion of a kinetoplast-associated gene attenuates virulence in Leishmania major parasites.},
journal = {Medical microbiology and immunology},
volume = {214},
number = {1},
pages = {16},
pmid = {40131505},
issn = {1432-1831},
mesh = {*Leishmania major/genetics/pathogenicity/immunology/growth & development ; Animals ; *CRISPR-Cas Systems ; *Leishmaniasis, Cutaneous/parasitology ; *Mice, Inbred BALB C ; Virulence ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Gene Deletion ; Female ; Cytokines/metabolism ; Macrophages/parasitology/immunology ; Disease Models, Animal ; DNA, Kinetoplast/genetics ; Gene Editing/methods ; Parasite Load ; },
abstract = {We employed a CRISPR/Cas9 technique in Leishmania major to evaluate its efficiency in editing a kDNA-associated gene, the universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. Using this toolkit, we generated UMSBP mNG-tagged and single knockout L. major (LmUMSBP[+/-]) parasites, which were confirmed by PCR, confocal microscopy and Western blot analyses. The growth rate of promastigotes in culture and their infectivity in macrophages were analysed in vitro. Mice were immunized with the LmUMSBP[+/-] mutant strain, and lesion size and parasite burden were measured upon challenge with wild-type (WT) L. major. Cytokines were quantified in supernatants of lymph node cell cultures. The results suggested successful expression and localization of the UMSBP mNG-tagged protein within the kinetoplast in both promastigote and intracellular amastigote forms, confirming the consistency of fluorescence tagging throughout various stages of the Leishmania life cycle. Attenuated LmUMSBP[+/-] parasites showed significantly reduced growth in culture (P < 0.05), increased apoptosis (P < 0.05) and downregulation of tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS) gene expression compared to WT L. major. LmUMSBP[+/-] mutant strains did not cause lesions in a susceptible BALB/c mouse model. Furthermore, immunization with LmUMSBP[+/-] parasites elicited a Th1 immune response, characterized by significantly higher IFN-γ and lower IL-4 production in cell culture (P < 0.001), which was associated with partial protection against WT L. major challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. In this study, we successfully validated a practical CRISPR/Cas9 toolkit in L. major, targeting the kinetoplast-associated gene UMSBP. Our findings suggest that the UMSBP single-allele knockout mutant holds promise as a valuable tool for studying the role of the kinetoplast in Leishmania biology and as a potential candidate for further investigation as a live-attenuated vaccine against Leishmania infection.},
}

*Leishmania major/genetics/pathogenicity/immunology/growth & development
Animals
*CRISPR-Cas Systems
*Leishmaniasis, Cutaneous/parasitology
*Mice, Inbred BALB C
Virulence
Mice
*Protozoan Proteins/genetics/metabolism
*Gene Deletion
Female
Cytokines/metabolism
Macrophages/parasitology/immunology
Disease Models, Animal
DNA, Kinetoplast/genetics
Gene Editing/methods
Parasite Load

@article {pmid40130324,
year = {2025},
author = {Wu, J and Meng, M and Guo, Z and Hao, K and Liang, Y and Meng, H and Fang, G and Shi, Z and Guo, X and Li, H and Feng, Y and Lin, L and Chen, J and Zhang, Y and Tian, H and Chen, X},
title = {Nuclear-Targeted Material Enabled Intranuclear MicroRNA Imaging for Tracking Gene Editing Process.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202500052},
doi = {10.1002/anie.202500052},
pmid = {40130324},
issn = {1521-3773},
abstract = {Gene editing technology based on clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas) systems serves as an efficient tool in cancer therapy. Tracking the gene editing process can help identify the progress of cancer treatment. However, existing techniques for monitoring the gene editing process rely on lysed cells, which can not reflect the dynamic changes of nucleic acid in living cells. It urgently needs in-situ and real-time imaging technologies to track gene editing process at living single cell level more effectively and precisely. Here, we reported a highly efficient nuclear-targeted material, phenylboronic acid modified linear PEI (LPBA), for loading gene editing plasmids and fluorescent probes to track gene editing processes of microRNA. Based on LPBA, we achieved efficient intranuclear microRNA imaging at the living cell level, reaching 32.4-fold higher than linear PEI (LPEI) delivery system, which facilitated further sensitive monitoring of the gene editing process both in living cells and in vivo. Meanwhile, this efficient gene-editing and real-time detection technique could be extended to screening effective gene editing plasmids. Such LPBA-based imaging technology extended imaging area of microRNA and offered new insight in the field of gene editing and nucleic acid detection.},
}

@article {pmid40130092,
year = {2025},
author = {Moradi, A and Khoshniyat, S and Nzeako, T and Khazeei Tabari, MA and Olanisa, OO and Tabbaa, K and Alkowati, H and Askarianfard, M and Daoud, D and Oyesanmi, O and Rodriguez, A and Lin, Y},
title = {The Future of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 Gene Therapy in Cardiomyopathies: A Review of Its Therapeutic Potential and Emerging Applications.},
journal = {Cureus},
volume = {17},
number = {2},
pages = {e79372},
pmid = {40130092},
issn = {2168-8184},
abstract = {Cardiomyopathies, among the leading causes of heart failure and sudden cardiac death, are often driven by genetic mutations affecting the heart's structural proteins. Despite significant advancements in understanding the genetic basis of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), effective long-term therapies remain limited. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing offers a promising therapeutic strategy to address these genetic disorders at their root. CRISPR-Cas9 enables precise modification of pathogenic variants (PVs) in genes encoding sarcomeric and desmosomal proteins, which are frequently implicated in cardiomyopathies. By inducing site-specific double-stranded breaks in DNA, followed by repair through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), this system allows for targeted correction of mutations. In preclinical models, CRISPR-Cas9 has shown promise in correcting HCM-associated mutations in β-myosin heavy chain 7 (MYH7), preventing disease phenotypes such as ventricular hypertrophy and myocardial fibrosis. Similarly, gene editing has successfully rectified DCM-linked mutations in Titin (TTN) and LMNA, resulting in improved heart function and reduced pathological remodeling. For ARVC, CRISPR-Cas9 has demonstrated the ability to repair mutations in desmosomal genes such as plakophilin 2 (PKP2), thereby restoring normal cardiac function and cellular adhesion. Despite these successes, challenges remain, including mosaicism, delivery efficiency, and off-target effects. Nevertheless, CRISPR-Cas9 represents a transformative approach to treating genetic cardiomyopathies, potentially offering long-lasting cures by directly addressing their underlying genetic causes.},
}

@article {pmid40129982,
year = {2025},
author = {Guo, Y and Zhu, M and Yu, Z and Li, Q and Chen, Y and Ci, L and Sun, R and Shen, R},
title = {Generation and characterization of a tamoxifen-inducible lineage tracing tool Cd2-P2A-CreERT2 knock-in mice.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1482070},
pmid = {40129982},
issn = {1664-3224},
mesh = {Animals ; *Tamoxifen/pharmacology ; Mice ; *Gene Knock-In Techniques ; *Integrases/genetics ; *CD2 Antigens/genetics ; *Mice, Transgenic ; *Cell Lineage/genetics ; CRISPR-Cas Systems ; Genes, Reporter ; Mice, Inbred C57BL ; },
abstract = {INTRODUCTION: The new targeted gene editing technologies, such as the CRISPR/Cas system, enable researchers to insert or delete genes at targeted loci efficiently. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity.

METHODS: Using the CRISPR/Cas9 system, we inserted the CreERT2 transgene expression cassette into the Cd2 gene locus to generate conditional Cre-driver line Cd2-CreERT2 knock-in mice, which drove the expression of CreERT2 by the endogenous Cd2 promoter. By mating the Cd2-CreERT2 strain with a Rosa26-LSL-tdTomato reporter mouse strain which contains a tdTomato expression fragment blocked with a loxP-flanked STOP cassette (LSL) driven by a CAG promoter, a Cd2-CreERT2;Rosa26-LSL-tdTomato reporter strain was obtained to evaluate the expression pattern of CD2 in different cell types.

RESULTS: After treatment with tamoxifen, the Cd2-CreERT2 knock-in mice were induced to perform efficient recombination at the loxP site following CreERT2 activation and cause the expression of tdTomato fluorescence. The tdTomato and CD2 were expressed in the T cells of peripheral blood, spleen and mesenteric lymph nodes, whereas detected in a low proportion in the B cells. While about 20% of cells labeled with tamoxifen-induced tdTomato were CD2[+] monocytes in peripheral blood, 10% of dendritic cells were tdTomato[+]/CD2[+] cells. Tamoxifen-independent expression of tdTomato occurred in approximately 3% of CD2[+] macrophages, but in negligible (~0.5%) in CD2[+] granulocytes.

DISCUSSION: This work supplied a new transgenic mouse as a valuable tool for lineage tracing in CD2-expressing cells, for conditional mutant studies of immune modulatory effects in a time-dependent manner, and analysis of the potential therapeutic effect of CD2-targeting biologics.},
}

Animals
*Tamoxifen/pharmacology
Mice
*Gene Knock-In Techniques
*Integrases/genetics
*CD2 Antigens/genetics
*Mice, Transgenic
*Cell Lineage/genetics
CRISPR-Cas Systems
Genes, Reporter
Mice, Inbred C57BL

@article {pmid40129518,
year = {2025},
author = {Chavhan, RL and Jaybhaye, SG and Hinge, VR and Deshmukh, AS and Shaikh, US and Jadhav, PK and Kadam, US and Hong, JC},
title = {Emerging applications of gene editing technologies for the development of climate-resilient crops.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1524767},
pmid = {40129518},
issn = {2673-3439},
abstract = {Climate change threatens global crop yield and food security due to rising temperatures, erratic rainfall, and increased abiotic stresses like drought, heat, and salinity. Gene editing technologies, including CRISPR/Cas9, base editors, and prime editors, offer precise tools for enhancing crop resilience. This review explores the mechanisms of these technologies and their applications in developing climate-resilient crops to address future challenges. While CRISPR/enables targeted modifications of plant DNA, the base editors allow for direct base conversion without inducing double-stranded breaks, and the prime editors enable precise insertions, deletions, and substitutions. By understanding and manipulating key regulator genes involved in stress responses, such as DREB, HSP, SOS, ERECTA, HsfA1, and NHX; crop tolerance can be enhanced against drought, heat, and salt stress. Gene editing can improve traits related to root development, water use efficiency, stress response pathways, heat shock response, photosynthesis, membrane stability, ion homeostasis, osmotic adjustment, and oxidative stress response. Advancements in gene editing technologies, integration with genomics, phenomics, artificial intelligence (AI)/machine learning (ML) hold great promise. However, challenges such as off-target effects, delivery methods, and regulatory barriers must be addressed. This review highlights the potential of gene editing to develop climate-resilient crops, contributing to food security and sustainable agriculture.},
}

@article {pmid40102632,
year = {2025},
author = {Salman, A and Song, WK and Storm, T and McClements, ME and MacLaren, RE},
title = {CRISPR targeting of SNPs associated with age-related macular degeneration in ARPE-19 cells: a potential model for manipulating the complement system.},
journal = {Gene therapy},
volume = {32},
number = {2},
pages = {132-141},
pmid = {40102632},
issn = {1476-5462},
mesh = {Humans ; *Macular Degeneration/genetics/therapy ; *Polymorphism, Single Nucleotide ; *Retinal Pigment Epithelium/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Cell Line ; Complement System Proteins/genetics ; Alleles ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Age-related Macular degeneration (AMD) is a major cause of vision loss and is linked to several predisposing single nucleotide polymorphisms (SNPs). CRISPR-mediated genome editing offers the potential to target negatively associated SNPs in an allele-specific manner, necessitating the need for a relevant cell model. The ARPE-19 cell line, with its stable monolayer growth and retinal pigment epithelium (RPE) characteristics, serves as an ideal model for AMD studies. Chronic inflammation and complement system dysregulation are implicated in AMD pathogenesis. Most genetic variations associated with AMD are in complement genes, suggesting their regulatory role. In this study, we conducted targeted PCRs to identify AMD-related SNPs in ARPE-19 cells and used CRISPR constructs to assess allele-specific activity. Guide RNA sequences were cloned into an EF-1-driven SpCas9 vector and packaged into lentivirus. Targeting efficiencies were evaluated with TIDE analysis, and allele-specificity was measured with NGS analysis 30 days post-transduction. Our results showed varying targeting efficiencies depending on guide RNA efficacy. For example, TIDE analysis of CFH SNPs rs1061170 and rs1410996 revealed efficiencies of 35.5% and 33.8%, respectively. CFB SNP rs4541862 showed efficiencies from 3% to 36.7%, and rs641153 ranged from 3.4% to 23.8%. Additionally, allele-specific targeting of AMD-related SNPs rs1061170, rs1410996, rs4541862, and rs641153 ranged from 48% to 52% in heterozygous differentiated ARPE-19 cells. These findings demonstrate the potential to manipulate the complement system in an AMD model by targeting disease-associated SNPs in an allele-specific manner, offering a promising therapeutic approach.},
}

Humans
*Macular Degeneration/genetics/therapy
*Polymorphism, Single Nucleotide
*Retinal Pigment Epithelium/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Cell Line
Complement System Proteins/genetics
Alleles
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40099511,
year = {2025},
author = {Wang, B and Yu, Z and Zhang, Z and Zhu, Z and Song, Y},
title = {A dCas9/sgRNA complex-mediated competitive assay for accurate and sensitive Pseudomonas aeruginosa analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {13},
pages = {2791-2798},
doi = {10.1039/d4ay02194k},
pmid = {40099511},
issn = {1759-9679},
mesh = {*Pseudomonas aeruginosa/genetics/isolation & purification ; CRISPR-Cas Systems ; Pseudomonas Infections/microbiology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Protein 9/genetics/chemistry ; DNA, Bacterial/analysis ; },
abstract = {Pseudomonas aeruginosa (P. aeruginosa), a Gram-negative pathogenic bacterium, is one of the most common bacteria that causes severe infectious diseases. However, accurate and efficient detection of P. aeruginosa in clinical samples is a huge challenge. Therefore, in this study, we developed a Cas9 derivative (dCas9)/sgRNA-mediated competitive assay for the sensitive and precise characterization of genomic materials from P. aeruginosa. Our approach involved the identification of target genomic sequences using the dCas9/sgRNA complex, which occupied the "sensing probe" (SP) binding site, resulting in an increased availability of free SP. SP subsequently facilitated DNA polymerase/endonuclease-mediated signal cycles and signal production, enabling highly sensitive detection of P. aeruginosa. The proposed competitive assay demonstrated a robust linear response to P. aeruginosa within a concentration range from 10 CFU mL[-1] to 10[6] CFU mL[-1], leveraging numerous signal amplification processes and competitive target recognition while exhibiting robust anti-interference capacity. Compared with former strategies, the proposed competitive assay enabled the accurate detection of P. aeruginosa by directly identifying and binding genomic sequences, which could be easily extended to the detection of other bacteria by simply changing the sgRNA. In addition, the proposed approach exhibits significant clinical potential for early disease diagnosis owing to its excellent sensitivity and accuracy.},
}

*Pseudomonas aeruginosa/genetics/isolation & purification
CRISPR-Cas Systems
Pseudomonas Infections/microbiology
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
CRISPR-Associated Protein 9/genetics/chemistry
DNA, Bacterial/analysis

@article {pmid40013946,
year = {2025},
author = {Scaramellini, N and Panzieri, DL and Cappellini, MD},
title = {Gene therapy for sickle cell disease and thalassemia.},
journal = {Current opinion in hematology},
volume = {32},
number = {3},
pages = {120-129},
doi = {10.1097/MOH.0000000000000867},
pmid = {40013946},
issn = {1531-7048},
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing ; *Thalassemia/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; Animals ; CRISPR-Cas Systems ; beta-Globins/genetics ; Genetic Vectors/therapeutic use/genetics ; },
abstract = {PURPOSE OF REVIEW: Thalassemia and sickle cell disease are among the most frequent monogenic hereditary diseases. Access to transfusions, iron chelation therapies and drugs such as hydroxyurea have improved life expectancy and quality of life. However, these diseases still cause significant disability. The first available curative therapy, bone marrow transplantation, is unfortunately not feasible for all patients. Over the past decade, numerous studies have focused on finding new curative therapies, and many clinical trials have evaluated different gene therapy approaches.

RECENT FINDINGS: The therapeutic targets focus on adding functional copies of the gene encoding β-globin in defective CD34 + cells, mainly using lentiviral vectors directed towards HSCs. More recently, the focus has shifted to inducing fetal hemoglobin production at therapeutic levels or repairing the underlying molecular defect, using novel gene editing techniques involving CRISPR-Cas9, transcription activation-like effector protein nucleases, zinc finger nucleases and base editing. Preclinical and clinical studies now focus on optimizing how gene therapy is performed and delivered to reduce or eliminate myeloablative treatment and its potential adverse events.

SUMMARY: In this review, we explore the potential to induce fetal hemoglobin production at therapeutic levels or to repair the underlying molecular defect that causes the disease genetically. Here, we review recent gene editing studies that are opening a new era in curative treatment for hemoglobinopathies.},
}

Humans
*Anemia, Sickle Cell/therapy/genetics
*Genetic Therapy/methods
*Gene Editing
*Thalassemia/therapy/genetics
Fetal Hemoglobin/genetics/biosynthesis
Animals
CRISPR-Cas Systems
beta-Globins/genetics
Genetic Vectors/therapeutic use/genetics

@article {pmid39948709,
year = {2025},
author = {Homchan, A and Patchsung, M and Chantanakool, P and Wongsatit, T and Onchan, W and Muengsaen, D and Thaweeskulchai, T and Tandean, M and Sakpetch, T and Suraritdechachai, S and Aphicho, K and Panchai, C and Taiwan, S and Horthongkham, N and Sudyoadsuk, T and Reinhardt, A and Uttamapinant, C},
title = {Recombinase-Controlled Multiphase Condensates Accelerate Nucleic Acid Amplification and CRISPR-Based Diagnostics.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {12},
pages = {10088-10103},
doi = {10.1021/jacs.4c11893},
pmid = {39948709},
issn = {1520-5126},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/chemistry/genetics ; CRISPR-Cas Systems/genetics ; Bacteriophage T4/enzymology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Isothermal techniques for amplifying nucleic acids have found extensive applications in genotyping and diagnostic tests. These methods can be integrated with sequence-specific detection strategies, such as CRISPR-based detection, for optimal diagnostic accuracy. In particular, recombinase-based amplification uses proteins from the Escherichia virus T4 recombination system and operates effectively at moderate temperatures in field and point-of-care settings. Here, we discover that recombinase polymerase amplification (RPA) is controlled by liquid-liquid phase separation, where the condensate formation enhances the nucleic acid amplification process. While two protein components of RPA could act as scaffold proteins for condensate formation, we identify T4 UvsX recombinase as the key regulator orchestrating distinct core-shell arrangements of proteins within multiphase condensates, with the intrinsically disordered C-terminus of UvsX being crucial for phase separation. We develop volumetric imaging assays to visualize RPA condensates and the reaction progression in whole volumes, and begin to dissect how macroscopic properties such as size distribution and droplet count could contribute to the overall reaction efficiency. Spatial organization of proteins in condensates may create optimal conditions for amplification, and disruption of such structures may diminish the amplification efficiency, as we demonstrate for the case of reverse transcription-RPA. The insight that RPA functions as a multiphase condensate leads us to identify the UvsX[D274A] mutant, which has a distinct phase-separation propensity compared to the wild-type enzyme and can enhance RNA detection via RPA-coupled CRISPR-based diagnostics.},
}

*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism/chemistry/genetics
CRISPR-Cas Systems/genetics
Bacteriophage T4/enzymology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39913998,
year = {2025},
author = {Tang, Y and Aleithan, F and Madahar, SS and Mirzaesmaeili, A and Saran, S and Tang, J and Zangiabadi, S and Inman, R and Sweeney, G and Abdul-Sater, AA},
title = {Selective disruption of Traf1/cIAP2 interaction attenuates inflammatory responses and rheumatoid arthritis.},
journal = {Journal of autoimmunity},
volume = {152},
number = {},
pages = {103377},
doi = {10.1016/j.jaut.2025.103377},
pmid = {39913998},
issn = {1095-9157},
mesh = {Animals ; *TNF Receptor-Associated Factor 1/metabolism/genetics ; *Arthritis, Rheumatoid/immunology/metabolism/genetics ; Mice ; Humans ; *Macrophages/immunology/metabolism ; Signal Transduction ; Inflammation/immunology/metabolism ; Baculoviral IAP Repeat-Containing 3 Protein/metabolism/genetics ; Arthritis, Experimental/immunology ; Disease Models, Animal ; NF-kappa B/metabolism ; Protein Binding ; Gene Editing ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: Tumor necrosis factor receptor-associated factor 1 (TRAF1) is an immune signaling adapter protein linked to increased susceptibility to rheumatoid arthritis (RA). TRAF1 has dual roles in regulating NF-κB and MAPK signaling: it promotes signaling through its association with cellular inhibitor of apoptosis 2 (cIAP2) downstream of certain tumor necrosis factor receptor (TNFR) family members but inhibits Toll-like receptor (TLR) signaling by limiting linear ubiquitination of key signaling proteins. In this study, we investigated whether selectively targeting TRAF1/cIAP2 interaction would lower inflammation and reduce severity of RA.

METHODS: We employed CRISPR/Cas9-mediated mediated gene editing to modify TRAF1 and specifically abrogate its interaction with cIAP2 in human macrophage cell lines and in mice. Biochemical studies were then employed to assess inflammatory signaling and cytokine production in gene edited macrophages. The collagen antibody-induced arthritis (CAIA) model of RA was used to trigger joint inflammation in mice.

RESULTS: We identify a critical mutation in TRAF1 (V203A in humans, V196A in mice) that disrupts its interaction with cIAP2, leading to a significant reduction in TLR signaling and downstream inflammation in human and murine macrophages. We demonstrate that TRAF1 is recruited to the TLR4 complex and is indispensable for the recruitment of cIAP2, facilitating TAK1 phosphorylation and the activation of NF-κB and MAPK signaling pathways. Remarkably, mice harboring the TRAF1 V196A mutation are protected from LPS-induced septic shock and exhibit markedly reduced joint inflammation and disease severity in the CAIA model of RA.

CONCLUSION: These findings reveal a previously unrecognized and crucial role for the TRAF1/cIAP2 axis in promoting inflammation and offer a promising foundation for the development of novel therapeutic strategies for inflammatory conditions, such as RA.},
}

Animals
*TNF Receptor-Associated Factor 1/metabolism/genetics
*Arthritis, Rheumatoid/immunology/metabolism/genetics
Mice
Humans
*Macrophages/immunology/metabolism
Signal Transduction
Inflammation/immunology/metabolism
Baculoviral IAP Repeat-Containing 3 Protein/metabolism/genetics
Arthritis, Experimental/immunology
Disease Models, Animal
NF-kappa B/metabolism
Protein Binding
Gene Editing
Cell Line
CRISPR-Cas Systems

@article {pmid39888280,
year = {2025},
author = {Rouatbi, N and Walters, AA and Zam, A and Lim, YM and Marrocu, A and Liam-Or, R and Anstee, JE and Arnold, JN and Wang, JT and Pollard, SM and Al-Jamal, KT},
title = {CD47 Knock-Out Using CRISPR-Cas9 RNA Lipid Nanocarriers Results in Reduced Mesenchymal Glioblastoma Growth In Vivo.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {12},
pages = {e2407262},
doi = {10.1002/advs.202407262},
pmid = {39888280},
issn = {2198-3844},
support = {CRI3645//Cancer Research Institute / Wade F.B. Thompson CLIP/ ; 202021-30//Brain Research UK/ ; GN-000398//Brain Tumour Charity/ ; MR/N013700/1/MRC_/Medical Research Council/United Kingdom ; DCRPGF∖100009/CRUK_/Cancer Research UK/United Kingdom ; WT103913/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*CD47 Antigen/genetics ; *Glioblastoma/genetics/therapy ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Nanoparticles/chemistry ; Disease Models, Animal ; Brain Neoplasms/genetics ; Lipids/chemistry ; Cell Line, Tumor ; Tumor Microenvironment/genetics/drug effects ; Liposomes ; },
abstract = {Immune checkpoint (ICP) blockade has shown limited effectiveness in glioblastoma (GBM), particularly in the mesenchymal subtype, where interactions between immune cells and glioblastoma cancer stem cells (GSCs) drive immunosuppression and therapy resistance. Tailoring ICPs specific to GSCs can enhance the antitumor immune response. This study proposes the use of lipid nanoparticles (LNPs) encapsulating CRISPR RNAs as an in vivo screening tool for ICPs in a syngeneic model of mesenchymal GSCs. Using PD-L1 and CD47 to validate the proof of concept, intratumoral administration of LNPs in orthotopic tumors achieved efficient editing of ICPs, leading to enhanced immune cell infiltration within the tumor microenvironment. Targeting CD47 reduced tumor growth, suggesting improved cancer cell sensitization to the immune system post-ICP editing. The study positions LNPs as a robust tool for in vivo validation of ICPs as therapeutic targets in clinically relevant GBM models. LNPs could serve as a screening tool in patient-derived xenografts to identify and optimize ICP combinations, potentially expediting ICP translation and enhancing personalized GBM immunotherapies.},
}

*CD47 Antigen/genetics
*Glioblastoma/genetics/therapy
Animals
Mice
*CRISPR-Cas Systems/genetics
Humans
*Nanoparticles/chemistry
Disease Models, Animal
Brain Neoplasms/genetics
Lipids/chemistry
Cell Line, Tumor
Tumor Microenvironment/genetics/drug effects
Liposomes

@article {pmid39870938,
year = {2025},
author = {Yan, W and Wang, S and Zhu, L and Yu, X and Li, J},
title = {Targeted editing of CCL5 with CRISPR-Cas9 nanoparticles enhances breast cancer immunotherapy.},
journal = {Apoptosis : an international journal on programmed cell death},
volume = {30},
number = {3-4},
pages = {912-935},
pmid = {39870938},
issn = {1573-675X},
mesh = {*Chemokine CCL5/genetics/metabolism/immunology ; *Breast Neoplasms/genetics/immunology/therapy/drug therapy ; Animals ; Female ; *CRISPR-Cas Systems/genetics ; Humans ; *Nanoparticles/chemistry ; Mice ; *Gene Editing/methods ; *Immunotherapy ; *Tumor Microenvironment/drug effects/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; },
abstract = {Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide. Immunotherapy, a promising therapeutic approach, often faces challenges due to the immunosuppressive tumor microenvironment. This study explores the innovative use of CRISPR-Cas9 technology in conjunction with FCPCV nanoparticles to target and edit the C-C Motif Chemokine Ligand 5 (CCL5) gene, aiming to improve the efficacy of breast cancer immunotherapy. Single-cell RNA sequencing (scRNA-seq) and TCGA-BRCA data identified CCL5 as a key immune-related gene in breast cancer. Using CRISPR-Cas9, sgRNA targeting CCL5 was designed and delivered to breast cancer cells and humanized mouse models via FCPCV nanoparticles. In vitro experiments demonstrated that FCPCV nanoparticles effectively silenced CCL5, enhanced CD8[+] T cell activity, and increased the production of cytokines such as IFN-γ, TNF-α, and GZMB. In vivo studies revealed significant tumor suppression, improved immune microenvironment, and increased CD8[+]/CD4[+] ratios in treated mice, without notable toxic side effects. These findings highlight the potential of CRISPR-Cas9 nanoparticle-mediated gene editing as a novel strategy for enhancing breast cancer immunotherapy, providing a new direction for personalized and effective cancer treatment.},
}

*Chemokine CCL5/genetics/metabolism/immunology
*Breast Neoplasms/genetics/immunology/therapy/drug therapy
Animals
Female
*CRISPR-Cas Systems/genetics
Humans
*Nanoparticles/chemistry
Mice
*Gene Editing/methods
*Immunotherapy
*Tumor Microenvironment/drug effects/immunology
CD8-Positive T-Lymphocytes/immunology
Cell Line, Tumor

@article {pmid39609594,
year = {2025},
author = {Murray, JB and Harrison, PT and Scholefield, J},
title = {Prime editing: therapeutic advances and mechanistic insights.},
journal = {Gene therapy},
volume = {32},
number = {2},
pages = {83-92},
pmid = {39609594},
issn = {1476-5462},
support = {20 FFP-P-8661//Science Foundation Ireland (SFI)/ ; DRUMM22G0-COLLAB//Cystic Fibrosis Foundation (CF Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; Animals ; },
abstract = {We are often confronted with a simple question, "which gene editing technique is the best?"; the simple answer is "there isn't one". In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, "classic" CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems
Animals

@article {pmid39587668,
year = {2025},
author = {Gong, X and Yan, Q and Chen, L},
title = {Transient receptor potential a1b regulates primordial germ cell numbers and sex differentiation in developing zebrafish.},
journal = {Journal of fish biology},
volume = {106},
number = {3},
pages = {921-931},
doi = {10.1111/jfb.16005},
pmid = {39587668},
issn = {1095-8649},
support = {32130109//the Key Project of the National Natural Science Foundation of China/ ; 2022YFD2400800//Key Technologies Research and Development Program/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Sex Differentiation/genetics ; *Zebrafish Proteins/genetics/metabolism ; Male ; *Germ Cells ; Female ; Sex Ratio ; TRPA1 Cation Channel/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Temperature is a leading environmental factor determining the sex ratio of some animal populations, such as fish, amphibians, and reptiles. However, the underlying mechanism by which temperature affects gender is still poorly understood. Transient receptor potential a1b (Trpa1b) belongs to the ion channel family of transient receptor potentials and exhibits dual thermosensitivity to heat and cold. In this study, we have unveiled a novel function of the trpa1b gene. Zebrafish generated through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with Trpa1b-null manifest a male-biased sex ratio. The quantity of primordial germ cells (PGCs) in zebrafish is closely linked to gender determination and gonadal development. Yet the role of the trpa1b gene in zebrafish reproductive development remains unexplored in the literature. Our investigation revealed a significant reduction in PGCs in Trpa1b mutant zebrafish compared to their wild-type counterparts 24-h postfertilization (hpf). Transcriptome sequencing of tissues near the reproductive crest of embryos at 1.25 days postfertilization (dpf) revealed differential changes in PGC-related marker genes and genes related to sperm cell development and differentiation. The relative expression of ddx4 and sycp3 genes was significantly downregulated, whereas amh was significantly upregulated at 20 dpf in trpa1b[-/-] zebrafish. The results of this study provide valuable insights and references for studying the molecular mechanism of sex determination in zebrafish. Undoubtedly, these results will further enhance our understanding of gender differentiation and gonadal development in fish and other vertebrates.},
}

Animals
*Zebrafish/genetics
*Sex Differentiation/genetics
*Zebrafish Proteins/genetics/metabolism
Male
*Germ Cells
Female
Sex Ratio
TRPA1 Cation Channel/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40128655,
year = {2025},
author = {Zhang, Y and Xiang, Y and Hou, D and Fang, L and Cai, S and Zhang, J and Wang, Y and Jiang, Y and Liu, B and Bai, J and Ding, Y and Fang, J and Chen, S and Liu, X and Ren, X},
title = {A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assay.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {163},
pmid = {40128655},
issn = {1471-2180},
support = {32400727//National Natural Science Foundation of China/ ; 24ZR1481100//Natural Science Foundation of Shanghai/ ; 2023YFC2307302//National Key Research and Development Program of China/ ; GWVI-11.2-XD29//program of Shanghai outstanding academic leader in public health subject/ ; 23141902300//experimental animal program sponsored by the Science and Technology Commission of Shanghai Municipality/ ; 22AH0901//Talent Project Foundation of PLA/ ; },
mesh = {*Dengue Virus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Dengue/diagnosis/virology ; *Sensitivity and Specificity ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Molecular Diagnostic Techniques/methods ; CRISPR-Associated Proteins/genetics ; RNA, Viral/genetics/isolation & purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Dengue Virus (DENV) is a life-threatening pathogen leading to dengue fever, which brings about huge public health challenges globally. However, traditional detection methods currently fail to meet the increasing demands of clinic practice in terms of speed, simplicity, and accuracy. To address these limitations, we developed a novel, rapid, and highly sensitive diagnostic method for universal DENV detection by integrating recombinase polymerase amplification (RPA) assay and the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system into one-pot. This approach achieves exceptional sensitivity and specificity for DENV detection, with the entire process completed within 40 min, without the need for sophisticated equipment. The limit of detection (LOD) was determined to be 91.7 copies/test. Using this one-pot RT-RPA CRISPR/Cas12a detection system, all four serotypes of DENV (1 to 4) were successfully identified. In terms of specificity, the assay accurately detected DENV-infected positive samples without cross-reactivity with four other interfering viruses-infected samples (VSV, SeV, HSV-1 and IAV). Furthermore, we established a universal DENV RT-RPA-CRISPR/Cas12a-lateral flow dipstick (LFD) platform, which successfully identified all four serotypes of DENV with a sensitivity of approximately 250 copies/test. Collectively, our method not only provides a robust alternative for universal DENV detection but also offers valuable insights for the identification of other viruses.},
}

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

@article {pmid40128351,
year = {2025},
author = {Ho Ching Chan, B and Hardy, H and Requena, T and Findlay, A and Ioannidis, J and Meunier, D and Toms, M and Moosajee, M and Raper, A and McGrew, MJ and Rainger, J},
title = {A stable NTN1 fluorescent reporter chicken reveals cell specific molecular signatures during optic fissure closure.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {10096},
pmid = {40128351},
issn = {2045-2322},
support = {BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BBS/E/D/10002071/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/S033165/1//UKRI Future Leaders Fellowship/ ; MR/S033165/1//UKRI Future Leaders Fellowship/ ; MR/S033165/1//UKRI Future Leaders Fellowship/ ; },
mesh = {Animals ; *Chickens ; Chick Embryo ; *Green Fluorescent Proteins/metabolism/genetics ; *Coloboma/genetics/metabolism/pathology ; *Netrin-1/metabolism/genetics ; *Genes, Reporter ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Eye/metabolism/embryology ; Neural Tube/metabolism/embryology ; Animals, Genetically Modified ; },
abstract = {NTN1 is expressed in a wide range of developmental tissues and is essential for normal development. Here we describe the generation of a Netrin-1 reporter chicken line (NTN1-T2A-eGFP) by targeting green fluorescent protein into the NTN1 locus using CRISPR/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (NTN1[T2A - eGFP/+]) embryos in which GFP localisation faithfully replicated endogenous NTN1 expression in the optic fissure and neural tube floorplate. Furthermore, all NTN1[T2A - eGFP/+] embryos and hatched birds appeared phenotypically normal. We applied this resource to a pertinent developmental context - coloboma is a structural eye malformation characterised by failure of epithelial fusion during optic fissure closure (OFC) and NTN1 is specifically expressed in fusion pioneer cells at the edges of the optic fissure. We therefore optimised the isolation of GFP expressing cells from embryonic NTN1[T2A - eGFP/+] eyes using spectral fluorescence cell-sorting and applied transcriptomic profiling of pioneer cells, which revealed multiple new OFC markers and novel pathways for developmental tissue fusion and coloboma. This work provides a novel fluorescent NTN1 chicken reporter line with broad experimental utility and is the first to directly molecularly characterise pioneer cells during OFC.},
}

Animals
*Chickens
Chick Embryo
*Green Fluorescent Proteins/metabolism/genetics
*Coloboma/genetics/metabolism/pathology
*Netrin-1/metabolism/genetics
*Genes, Reporter
CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Eye/metabolism/embryology
Neural Tube/metabolism/embryology
Animals, Genetically Modified

@article {pmid40127793,
year = {2025},
author = {Yan, C and Zhang, Z and Xu, J and Meng, Y and Yan, S and Wei, L and Zou, Q and Zhang, Q and Cui, F},
title = {CasPro-ESM2: Accurate identification of Cas proteins integrating pre-trained protein language model and multi-scale convolutional neural network.},
journal = {International journal of biological macromolecules},
volume = {308},
number = {Pt 1},
pages = {142309},
doi = {10.1016/j.ijbiomac.2025.142309},
pmid = {40127793},
issn = {1879-0003},
abstract = {Cas proteins (CRISPR-associated protein) are the core components of the CRISPR-Cas system, playing critical roles in defending against foreign DNA and RNA invasions. Identifying Cas proteins can provide deeper insights into the immune mechanisms of the CRISPR-Cas system and help uncover the functional mechanisms of Cas proteins. In this study, we developed a computational tool named CasPro-ESM2, which combines the Pre-trained Protein Language Model ESM-2, multi-scale convolutional neural networks, and evolutionary information from protein sequences to identify Cas proteins. Experimental results demonstrate that CasPro-ESM2 outperforms existing models in Cas protein identification, achieving the highest values in metrics such as ACC, SP, SN, and MCC on two different datasets. Furthermore, we deployed this tool on a web server to enable direct access for users (http://www.bioai-lab.com/CasProESM-2).},
}

@article {pmid40126363,
year = {2025},
author = {Griffin, C and Coppenrath, K and Khan, D and Lin, Z and Horb, M and Saint-Jeannet, JP},
title = {Deletion of sf3b4 causes splicing defects and gene dysregulation that disrupt craniofacial development and survival.},
journal = {Disease models & mechanisms},
volume = {18},
number = {3},
pages = {},
doi = {10.1242/dmm.052169},
pmid = {40126363},
issn = {1754-8411},
support = {R24-OD030008//National Institutes of Health office of the Director/ ; R01-DE025468/DE/NIDCR NIH HHS/United States ; //New York University/ ; },
mesh = {Animals ; *Neural Crest/pathology/metabolism ; *RNA Splicing/genetics ; *RNA Splicing Factors/genetics/metabolism ; Xenopus/embryology ; Skull/embryology/pathology ; Gene Deletion ; Xenopus Proteins/genetics/metabolism ; Mandibulofacial Dysostosis/genetics/pathology ; Gene Expression Regulation, Developmental ; Exons/genetics ; Cartilage/pathology/embryology ; Craniofacial Abnormalities/genetics/pathology ; Sequence Deletion ; Cell Movement/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Nager and Rodriguez syndromes are rare craniofacial and limb disorders characterized by midface retrusion, micrognathia, absent thumbs and radial hypoplasia. These disorders result from haploinsufficiency of SF3B4 (splicing factor 3b, subunit 4), a component of the pre-mRNA spliceosomal machinery. Although the spliceosome is present and functions in all cells of the body, most spliceosomopathies - including Nager and Rodriguez syndromes - are cell- or tissue-specific in their pathology. To understand the pathomechanism underlying these conditions, we generated a Xenopus tropicalis sf3b4 mutant line using CRISPR/Cas9 gene-editing technology. Homozygous deletion of sf3b4 is detrimental to the development of cranial neural crest (NC)-derived cartilage progenitors. Temporal RNA-sequencing analyses of mutant embryos identified an increase in exon-skipping events, followed by important transcriptional changes associated with an enrichment for terms consistent with defects in NC cell migration and survival. We propose that disruption of these processes may underly the pathogenesis of Nager and Rodriguez syndromes.},
}

Animals
*Neural Crest/pathology/metabolism
*RNA Splicing/genetics
*RNA Splicing Factors/genetics/metabolism
Xenopus/embryology
Skull/embryology/pathology
Gene Deletion
Xenopus Proteins/genetics/metabolism
Mandibulofacial Dysostosis/genetics/pathology
Gene Expression Regulation, Developmental
Exons/genetics
Cartilage/pathology/embryology
Craniofacial Abnormalities/genetics/pathology
Sequence Deletion
Cell Movement/genetics
CRISPR-Cas Systems/genetics

@article {pmid40125908,
year = {2025},
author = {Niu, H and Zou, L and Liu, Y and Li, Z and Ren, H and Liao, H and Zhang, X and An, S and Ren, F and Ge, X and Cheng, L and Yang, F and Pan, H and Rong, S and Chang, D and Ma, H},
title = {CRISPR/Cas System-Based Fluorescent Sensor for Analysis and Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-16},
doi = {10.1080/10408347.2025.2481409},
pmid = {40125908},
issn = {1547-6510},
abstract = {Fluorescent sensor is an important tool to reliaze qualitative or quantitative detection of target analyte based on the fluorescence principle. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has been utilized to develop as a precise, efficient, and highly sensitive molecular diagnostic tool due to its efficient targeting and gene editing ability. At present, CRISPR/Cas system-based fluorescent sensors have shown excellent performance in the field of analysis and detection, and have received widespread attention. Therefore, this paper reviews the mechanism of the CRISPR/Cas system, the characteristics of different Cas proteins, and the principle and characteristics of the fluorescent sensor, with a focus on summarizing the application of the CRISPR/Cas system-based fluorescent sensor for analysis and detection.},
}

@article {pmid40125088,
year = {2025},
author = {Cao, ML and Han, RY and Chen, SD and Zhao, DY and Shi, MY and Zou, JH and Li, L and Jiang, HK},
title = {Gene Editing: An Effective Tool for the Future Treatment of Kidney Disease.},
journal = {Journal of inflammation research},
volume = {18},
number = {},
pages = {4001-4018},
pmid = {40125088},
issn = {1178-7031},
abstract = {Gene editing technology involves modifying target genes to alter genetic traits and generate new phenotypes. Beginning with zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), the field has evolved through the advent of clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems, and more recently to base editors (BE) and prime editors (PE). These innovations have provided deep insights into the molecular mechanisms of complex biological processes and have paved the way for novel therapeutic strategies for a range of diseases. Gene editing is now being applied in the treatment of both genetic and acquired kidney diseases, as well as in kidney transplantation and the correction of genetic mutations. This review explores the current applications of mainstream gene editing technologies in biology, with a particular emphasis on their roles in kidney disease research and treatment of. It also addresses the limitations and challenges associated with these technologies, while offering perspectives on their future potential in this field.},
}

@article {pmid40123515,
year = {2025},
author = {Hu, C and Shi, X and Guo, B and Yang, Z and Zhou, J and Wang, F},
title = {Toehold-Based CRISPR-dCas9 Transcriptional Activation Platform for Spatiotemporally Controllable Gene Therapy in Tumor and Diabetic Mouse Models.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c01078},
pmid = {40123515},
issn = {1936-086X},
abstract = {The CRISPR-Cas system has been extensively employed as a genome editing tool with the dCas9-based transcriptional activation system emerging as a particularly promising approach for gene editing in the treatment of diseases at the gene level. Nevertheless, the challenge of achieving effective spatiotemporal control of the transcriptional activation system of dCas9 has thus far restricted its broader application. In this study, we present an miRNA-responsive CRISPR-dCas9 transcriptional activation (mCTA) system. This system is capable of responding specifically to exogenous and endogenous miRNAs in mammalian cells and enables the specific imaging of miRNAs during neural development or in the deep tissues of mice. Furthermore, the replacement of downstream functional genes with DTA has been demonstrated to result in the effective apoptosis of tumor cells and inhibition of xenografted tumor growth in mice. Finally, in a diabetic mouse model, the m122CTA system was shown to reduce the blood glucose in diabetic mice via the activation of PDX-1 gene. Our work provides an effective platform for miRNA imaging and gene therapy via spatiotemporal control of gene regulation.},
}

@article {pmid40121659,
year = {2025},
author = {Xu, P and Huang, J and Chen, X and Wang, Q and Yin, B and Xian, Q and Zhuang, C and Hu, Y},
title = {Efficient targeted T-DNA integration for gene activation and male germline-specific gene tagging in Arabidopsis.},
journal = {The Plant journal : for cell and molecular biology},
volume = {121},
number = {6},
pages = {e70104},
doi = {10.1111/tpj.70104},
pmid = {40121659},
issn = {1365-313X},
support = {2023A1515010817//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 31771342//National Natural Science Foundation of China/ ; },
mesh = {*Arabidopsis/genetics ; *DNA, Bacterial/genetics ; *Arabidopsis Proteins/genetics/metabolism ; Plants, Genetically Modified ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; DNA End-Joining Repair/genetics ; Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Transcriptional Activation/genetics ; },
abstract = {Site-specific DNA integration is an important tool in plant genetic engineering. Traditionally, this process relies on homologous recombination (HR), which is known for its low efficiency in plant cells. In contrast, Agrobacterium-mediated T-DNA integration is highly efficient for plant transformation. However, T-DNA is typically inserted randomly into double-strand breaks within the plant genome via the non-homologous end-joining (NHEJ) DNA repair pathway. In this study, we developed an approach of CRISPR/Cas9-mediated targeted T-DNA integration in Arabidopsis, which was more rapid and efficient than the HR-mediated method. This targeted T-DNA integration aided in gene activation and male germline-specific gene tagging. Gene activation was accomplished by positioning the CaMV35S promoter at the left border of T-DNA, thereby activating specific downstream genes. The activation of FT and MYB26 significantly increased their transcriptional expression, which resulted in early flowering and an altered pattern of cell wall thickening in the anther endothelium, respectively. Male germline-specific gene tagging incorporates two reporters, namely, NeoR and MGH3::mCherry, within the T-DNA. This design facilitates the creation of insertional mutants, simplifies the genetic analysis of mutated alleles, and allows for cellular tracking of male germline cells during fertilization. We successfully applied this system to target the male germline-specific gene GEX2. In conclusion, our results demonstrated that site-specific integration of DNA fragments in the plant genome can be rapidly and efficiently achieved through the NHEJ pathway, making this approach broadly applicable in various contexts.},
}

*Arabidopsis/genetics
*DNA, Bacterial/genetics
*Arabidopsis Proteins/genetics/metabolism
Plants, Genetically Modified
CRISPR-Cas Systems
Gene Expression Regulation, Plant
DNA End-Joining Repair/genetics
Transcription Factors/genetics/metabolism
Promoter Regions, Genetic/genetics
Transcriptional Activation/genetics

@article {pmid40121601,
year = {2025},
author = {Vollen, K and Alonso, JM and Stepanova, AN},
title = {Beyond a few bases: methods for large DNA insertion and gene targeting in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {121},
number = {6},
pages = {e70099},
pmid = {40121601},
issn = {1365-313X},
support = {1444561//National Science Foundation/ ; 1750006//National Science Foundation/ ; 1940829//National Science Foundation/ ; 2023356574//National Science Foundation/ ; 2327912//National Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Targeting/methods ; *Crops, Agricultural/genetics ; Plants/genetics ; DNA, Plant/genetics ; Mutagenesis, Insertional ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; },
abstract = {Genome editing technologies like CRISPR/Cas have greatly accelerated the pace of both fundamental research and translational applications in agriculture. However, many plant biologists are functionally limited to creating small, targeted DNA changes or large, random DNA insertions. The ability to efficiently generate large, yet precise, DNA changes will massively accelerate crop breeding cycles, enabling researchers to more efficiently engineer crops amidst a rapidly changing agricultural landscape. This review provides an overview of existing technologies that allow plant biologists to integrate large DNA sequences within a plant host and some associated technical bottlenecks. Additionally, this review explores a selection of emerging techniques in other host systems to inspire tool development in plants.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Targeting/methods
*Crops, Agricultural/genetics
Plants/genetics
DNA, Plant/genetics
Mutagenesis, Insertional
Genome, Plant/genetics
Plants, Genetically Modified/genetics
Plant Breeding/methods

@article {pmid39962231,
year = {2025},
author = {Zakrzewski, P and Rice, CM and Fleming, K and Cela, D and Groves, SJ and Ponce-Garcia, FM and Gibbs, W and Roberts, K and Pike, T and Strathdee, D and Anderson, E and Nobbs, AH and Toye, AM and Steward, C and Amulic, B},
title = {Tafazzin regulates neutrophil maturation and inflammatory response.},
journal = {EMBO reports},
volume = {26},
number = {6},
pages = {1590-1619},
pmid = {39962231},
issn = {1469-3178},
support = {MR/R02149X/1//Medical Research Foundation (MRF)/ ; WP15-05//NHS Blood and Transplant R&D grant/ ; IS-BTU-1214-10032//NIHR BTRU in partnership with NHSBT/ ; A31287//CRUK Scotland Institute core funding/ ; },
mesh = {Humans ; *Neutrophils/metabolism/immunology ; *Neutropenia/genetics/congenital ; *Unfolded Protein Response ; *Cell Differentiation ; Inflammation/genetics/metabolism/pathology ; Extracellular Traps/metabolism/genetics ; CRISPR-Cas Systems ; Hematopoietic Stem Cells/metabolism ; Gene Editing ; Congenital Bone Marrow Failure Syndromes/genetics/pathology/metabolism ; Transcription Factors/metabolism/genetics ; Mutation ; Signal Transduction ; Cell Degranulation ; },
abstract = {Barth syndrome (BTHS) is a rare genetic disease caused by mutations in the TAFAZZIN gene. It is characterized by neutropenia, cardiomyopathy and skeletal myopathy. Neutropenia in BTHS is associated with life-threatening infections, yet there is little understanding of the molecular and physiological causes of this phenomenon. We combined bone marrow analysis, CRISPR/Cas9 genome editing in hematopoietic stem cells and functional characterization of circulating BTHS patient neutrophils to investigate the role of TAFAZZIN in neutrophils and their progenitors. We demonstrate a partial cell intrinsic differentiation defect, along with a dysregulated neutrophil inflammatory response in BTHS, including elevated degranulation and formation of neutrophil extracellular traps (NETs) in response to calcium flux. Developmental and functional alterations in BTHS neutrophils are underpinned by perturbations in the unfolded protein response (UPR) signaling pathway, suggesting potential therapeutic avenues for targeting BTHS neutropenia.},
}

Humans
*Neutrophils/metabolism/immunology
*Neutropenia/genetics/congenital
*Unfolded Protein Response
*Cell Differentiation
Inflammation/genetics/metabolism/pathology
Extracellular Traps/metabolism/genetics
CRISPR-Cas Systems
Hematopoietic Stem Cells/metabolism
Gene Editing
Congenital Bone Marrow Failure Syndromes/genetics/pathology/metabolism
Transcription Factors/metabolism/genetics
Mutation
Signal Transduction
Cell Degranulation

@article {pmid39930152,
year = {2025},
author = {Zhang, P and Zhang, Z and Wang, Y and Du, W and Song, X and Lai, W and Wang, H and Zhu, B and Xiong, J},
title = {A CRISPR-Cas9 screen reveals genetic determinants of the cellular response to decitabine.},
journal = {EMBO reports},
volume = {26},
number = {6},
pages = {1528-1565},
pmid = {39930152},
issn = {1469-3178},
support = {32288102//MOST | National Natural Science Foundation of China (NSFC)/ ; 32170607//MOST | National Natural Science Foundation of China (NSFC)/ ; 32000417//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFC2300500//MOST | National Key Research and Development Program of China (NKPs)/ ; 2017133//CAS | Youth Innovation Promotion Association (YIPA)/ ; 2020097//CAS | Youth Innovation Promotion Association (YIPA)/ ; },
mesh = {*Decitabine/pharmacology ; Humans ; *CRISPR-Cas Systems ; *DNA Damage ; Histone Demethylases/metabolism/genetics ; DNA Methylation/drug effects ; Leukemia, Myeloid, Acute/genetics/drug therapy/pathology ; Antimetabolites, Antineoplastic/pharmacology ; DNA Repair/drug effects ; DNA Replication/drug effects ; Cell Line, Tumor ; },
abstract = {Decitabine (DAC), a well-recognized DNA hypomethylating agent, has been applied to treat acute myeloid leukemia. However, clinic investigations revealed that DNA methylation reduction does not correlate with a clinical response, and relapse is prevalent. To gain a better understanding of its anti-tumor mechanism, we perform a temporally resolved CRISPR-Cas9 screen to identify factors governing the DAC response. We show that DNA damage generated by DNMT-DNA adducts and 5-aza-dUTP misincorporation through the dCMP deaminase DCTD act as drivers of DAC-induced acute cytotoxicity. The DNA damage that arises during the next S phase is dependent on DNA replication, unveiling a trans-cell cycle effect of DAC on genome stability. By exploring candidates for synthetic lethality, we unexpectedly uncover that KDM1A promotes survival after DAC treatment through interactions with ZMYM3 and CoREST, independent of its demethylase activity or regulation of viral mimicry. These findings emphasize the importance of DNA repair pathways in DAC response and provide potential biomarkers.},
}

*Decitabine/pharmacology
Humans
*CRISPR-Cas Systems
*DNA Damage
Histone Demethylases/metabolism/genetics
DNA Methylation/drug effects
Leukemia, Myeloid, Acute/genetics/drug therapy/pathology
Antimetabolites, Antineoplastic/pharmacology
DNA Repair/drug effects
DNA Replication/drug effects
Cell Line, Tumor

@article {pmid39918307,
year = {2025},
author = {Dougherty, MW and Hoffmann, RM and Hernandez, MC and Airan, Y and Gharaibeh, RZ and Herzon, SB and Yang, Y and Jobin, C},
title = {Genome-scale CRISPR/Cas9 screening reveals the role of PSMD4 in colibactin-mediated cell cycle arrest.},
journal = {mSphere},
volume = {10},
number = {3},
pages = {e0069224},
doi = {10.1128/msphere.00692-24},
pmid = {39918307},
issn = {2379-5042},
support = {R01CA215553//HHS | National Institutes of Health (NIH)/ ; //University of Florida Health Cancer Center/ ; //University of Florida Gatorade Fund/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Polyketides/metabolism ; *Peptides/metabolism/genetics ; *Cell Cycle Checkpoints/genetics ; HT29 Cells ; HEK293 Cells ; Cell Survival ; Gene Knockout Techniques ; DNA Damage ; },
abstract = {UNLABELLED: Colibactin is a genotoxic secondary metabolite produced by certain Enterobacteriaceae strains that populate the intestine and produces a specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed genome-wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks[+] bacteria and a synthetic colibactin analog. We identified 20 enriched genes with a MAGeCK score of >2.0 in both screens, including proteasomal subunits (e.g., PSMG4 and PSMD4), RNA processing factors (e.g., SF1 and PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout in HEK293T and HT-29 cells promoted cell viability and ameliorated G2-M cell cycle arrest but did not affect the amount of phosphorylated H2AX foci after exposure to synthetic colibactin 742. Consistent with these observations, PSMD4[-/-] cells had a significantly higher colony formation rate and bigger colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell cycle arrest following colibactin-induced DNA damage and that cells with PSMD4 deficiency may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.

IMPORTANCE: Colibactin has been implicated as a causative agent of colorectal cancer. However, colibactin-producing bacteria are also present in many healthy individuals, leading to the hypothesis that some aspects of colibactin regulation or host response dictate the molecule's carcinogenic potential. Elucidating the host-response pathways involved in dictating cell fate after colibactin intoxication has been difficult, partially due to an inability to isolate the molecule. This study provides the first high-throughput CRISPR/Cas9 screening to identify genes conferring colibactin sensitivity. Here, we utilize both bacterial infection and a synthetic colibactin analog to identify genes directly involved in colibactin response. These findings provide insight into how differences in gene expression may render certain individuals more vulnerable to colibactin-initiated tumor formation after DNA damage.},
}

Humans
*CRISPR-Cas Systems
*Polyketides/metabolism
*Peptides/metabolism/genetics
*Cell Cycle Checkpoints/genetics
HT29 Cells
HEK293 Cells
Cell Survival
Gene Knockout Techniques
DNA Damage

@article {pmid39887606,
year = {2025},
author = {Li, Z and Kim, M and da Silva Nascimento, JR and Legeret, B and Jorge, GL and Bertrand, M and Beisson, F and Thelen, JJ and Li-Beisson, Y},
title = {Knocking out the carboxyltransferase interactor 1 (CTI1) in Chlamydomonas boosted oil content by fivefold without affecting cell growth.},
journal = {Plant biotechnology journal},
volume = {23},
number = {4},
pages = {1230-1242},
doi = {10.1111/pbi.14581},
pmid = {39887606},
issn = {1467-7652},
support = {IOS-1829365//National Science Foundation/ ; DE-SC0023142//Department of Energy Office of Biological and Environmental Research (BER)/ ; //CEA 'Circular Carbon Economy'/ ; },
mesh = {*Chlamydomonas/genetics/metabolism/growth & development/enzymology ; Fatty Acids/metabolism ; Chlamydomonas reinhardtii/genetics/metabolism/growth & development/enzymology ; Plant Proteins/genetics/metabolism ; Acetyl-CoA Carboxylase/metabolism/genetics ; Triglycerides/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; },
abstract = {The first step in chloroplast de novo fatty acid synthesis is catalysed by acetyl-CoA carboxylase (ACCase). As the rate-limiting step for this pathway, ACCase is subject to both positive and negative regulation. In this study, we identify a Chlamydomonas homologue of the plant carboxyltransferase interactor 1 (CrCTI1) and show that this protein interacts with the Chlamydomonas α-carboxyltransferase (Crα-CT) subunit of the ACCase by yeast two-hybrid protein-protein interaction assay. Three independent CRISPR-Cas9 mediated knockout mutants for CrCTI1 each produced an 'enhanced oil' phenotype, accumulating 25% more total fatty acids and storing up to fivefold more triacylglycerols (TAGs) in lipid droplets. The TAG phenotype of the crcti1 mutants was not influenced by light but was affected by trophic growth conditions. By growing cells under heterotrophic conditions, we observed a crucial function of CrCTI1 in balancing lipid accumulation and cell growth. Mutating a previously mapped in vivo phosphorylation site (CrCTI1 Ser108 to either Ala or to Asp), did not affect the interaction with Crα-CT. However, mutating all six predicted phosphorylation sites within Crα-CT to create a phosphomimetic mutant reduced this pairwise interaction significantly. Comparative proteomic analyses of the crcti1 mutants and WT suggested a role for CrCTI1 in regulating carbon flux by coordinating carbon metabolism, antioxidant and fatty acid β-oxidation pathways, to enable cells to adapt to carbon availability. Taken together, this study identifies CrCTI1 as a negative regulator of fatty acid synthesis in algae and provides a new molecular brick for the genetic engineering of microalgae for biotechnology purposes.},
}

*Chlamydomonas/genetics/metabolism/growth & development/enzymology
Fatty Acids/metabolism
Chlamydomonas reinhardtii/genetics/metabolism/growth & development/enzymology
Plant Proteins/genetics/metabolism
Acetyl-CoA Carboxylase/metabolism/genetics
Triglycerides/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems/genetics

@article {pmid39846980,
year = {2025},
author = {Xin, H and Strickland, LW and Hamilton, JP and Trusky, JK and Fang, C and Butler, NM and Douches, DS and Buell, CR and Jiang, J},
title = {Jan and mini-Jan, a model system for potato functional genomics.},
journal = {Plant biotechnology journal},
volume = {23},
number = {4},
pages = {1243-1256},
doi = {10.1111/pbi.14582},
pmid = {39846980},
issn = {1467-7652},
support = {IS-5317-20C//United States - Israel Binational Agricultural Research and Development Fund/ ; IS-5684-24C//United States - Israel Binational Agricultural Research and Development Fund/ ; T32GM110523/GM/NIGMS NIH HHS/United States ; T32GM152798/GM/NIGMS NIH HHS/United States ; T32GM110523/GM/NIGMS NIH HHS/United States ; T32GM152798/GM/NIGMS NIH HHS/United States ; },
mesh = {*Solanum tuberosum/genetics/growth & development ; *Genomics ; *Genome, Plant/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Potato (Solanum tuberosum) is the third-most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags behind that of other major food crops, largely due to the lack of a model experimental potato line. Here, we present a diploid potato line, 'Jan,' which possesses all essential characteristics for facile functional genomics studies. Jan exhibits a high level of homozygosity after seven generations of self-pollination. Jan is vigorous, highly fertile and produces tubers with outstanding traits. Additionally, it demonstrates high regeneration rates and excellent transformation efficiencies. We generated a chromosome-scale genome assembly for Jan, annotated its genes and identified syntelogs relative to the potato reference genome assembly DMv6.1 to facilitate functional genomics. To miniaturize plant architecture, we developed two 'mini-Jan' lines with compact and dwarf plant stature through CRISPR/Cas9-mediated mutagenesis targeting the Dwarf and Erecta genes involved in growth. One mini-Jan mutant, mini-Jan[E], is fully fertile and will permit higher-throughput studies in limited growth chamber and greenhouse space. Thus, Jan and mini-Jan offer a robust model system that can be leveraged for gene editing and functional genomics research in potato.},
}

*Solanum tuberosum/genetics/growth & development
*Genomics
*Genome, Plant/genetics
CRISPR-Cas Systems/genetics
Gene Editing/methods
Plants, Genetically Modified/genetics

@article {pmid39799585,
year = {2025},
author = {Bai, S and Cao, X and Hu, L and Hu, D and Li, D and Sun, Y},
title = {Engineering an optimized hypercompact CRISPR/Cas12j-8 system for efficient genome editing in plants.},
journal = {Plant biotechnology journal},
volume = {23},
number = {4},
pages = {1153-1164},
doi = {10.1111/pbi.14574},
pmid = {39799585},
issn = {1467-7652},
support = {31800206//National Natural Science Foundation of China/ ; 32160111//National Natural Science Foundation of China/ ; 32370431//National Natural Science Foundation of China/ ; NJYT22105//Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region/ ; 2020MS03027//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2021ZD04//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2020ZY0005//Central Government Guiding Special Funds for the Development of Local Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Oryza/genetics ; Genome, Plant/genetics ; Glycine max/genetics ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; },
abstract = {The Cas12j-8 nuclease, derived from the type V CRISPR system, is approximately half the size of Cas9 and recognizes a 5'-TTN-3' protospacer adjacent motif sequence, thus potentially having broad application in genome editing for crop improvement. However, its editing efficiency remains low in plants. In this study, we rationally engineered both the crRNA and the Cas12j-8 nuclease. The engineered crRNA and Cas12j-8 markedly improved genome editing efficiency in plants. When combined, they exhibited robust editing activity in soybean and rice, enabling the editing of target sites that were previously uneditable. Notably, for certain target sequences, the editing activity was comparable to that of SpCas9 when targeting identical sequences, and it outperformed the Cas12j-2 variant, nCas12j-2, across all tested targets. Additionally, we developed cytosine base editors based on the engineered crRNA and Cas12j-8, demonstrating an average increase of 5.36- to 6.85-fold in base-editing efficiency (C to T) compared with the unengineered system in plants, with no insertions or deletions (indels) observed. Collectively, these findings indicate that the engineered hypercompact CRISPR/Cas12j-8 system serves as an efficient tool for genome editing mediated by both nuclease cleavage and base editing in plants.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Oryza/genetics
Genome, Plant/genetics
Glycine max/genetics
Plants, Genetically Modified/genetics
Genetic Engineering/methods

@article {pmid39799577,
year = {2025},
author = {Wei, XY and Pei, Y and Liu, L and Hamar, P and Pei, DS},
title = {Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology.},
journal = {STAR protocols},
volume = {6},
number = {1},
pages = {103544},
pmid = {39799577},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Fishes/genetics ; *DNA, Environmental/genetics/analysis/isolation & purification ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {The recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system enables sensitive detection of environmental DNA (eDNA) in rare fish species. Here, we present a protocol for eDNA amplification and Cas12a for target recognition using RPA. We describe steps for identifying a target site, synthesis and purification of CRISPR RNA (crRNA), and RPA isothermal amplification. We then detail procedures for constructing the eDNA CRISPR-Cas12a detection system and verifying its sensitivity. This protocol offers a high-sensitivity approach for monitoring aquatic biodiversity and conservation efforts, even in low eDNA concentrations. For complete details on the use and execution of this protocol, please refer to Wei et al.[1].},
}

Animals
*CRISPR-Cas Systems/genetics
*Fishes/genetics
*DNA, Environmental/genetics/analysis/isolation & purification
Nucleic Acid Amplification Techniques/methods

@article {pmid39756031,
year = {2025},
author = {Teng, Y and Arbogast, K and Junge, H and Chen, Z},
title = {Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter.},
journal = {STAR protocols},
volume = {6},
number = {1},
pages = {103543},
pmid = {39756031},
issn = {2666-1667},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Mice, Transgenic ; Mutation/genetics ; Alternative Splicing/genetics ; Genes, Reporter/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA Splicing/genetics ; Protein Isoforms/genetics ; },
abstract = {Here, we present a protocol to alter the production of alternatively spliced mRNA variants, without affecting the overall gene expression, through CRISPR-Cas9-engineered genomic mutations in mice. We describe steps for designing guide RNA to direct Cas9 endonuclease to consensus splice sites, producing transgenic mice through pronuclear injection, and screening for desired mutations in cultured mammalian cells using a minigene splicing reporter. Splice isoform-specific mouse mutants provide valuable tools for genetic analyses beyond loss-of-function and transgenic alleles. For complete details on the use and execution of this protocol, please refer to Dailey-Krempel et al.[1] and Johnson et al.[2].},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
*Mice, Transgenic
Mutation/genetics
Alternative Splicing/genetics
Genes, Reporter/genetics
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
RNA Splicing/genetics
Protein Isoforms/genetics

@article {pmid39709609,
year = {2025},
author = {Hu, Y and Chen, Y and Zhang, Y and Liu, Z and Li, J},
title = {Protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system.},
journal = {STAR protocols},
volume = {6},
number = {1},
pages = {103516},
pmid = {39709609},
issn = {2666-1667},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; *Germ Cells/metabolism/cytology ; *DNA Transposable Elements/genetics ; *Plasmids/genetics ; *Mutagenesis/genetics ; Microinjections/methods ; Animals, Genetically Modified ; },
abstract = {Here, we present a protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid system. We describe steps for conditional mutagenesis plasmid construction, zebrafish embryo microinjection, and screening for green fluorescence in the heart. This protocol is simple to execute, time efficient, and multifunctional, enabling the disruption of genes in zebrafish germ cells to be conducted with ease. For complete details on the use and execution of this protocol, please refer to Hu et al.[1].},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems/genetics
*Germ Cells/metabolism/cytology
*DNA Transposable Elements/genetics
*Plasmids/genetics
*Mutagenesis/genetics
Microinjections/methods
Animals, Genetically Modified

@article {pmid39700011,
year = {2025},
author = {Benz, T and Larghero, P and Meyer, C and Müller, M and Brüggmann, D and Hentrich, AE and Louwen, F and Erkner, E and Fitzel, R and Schneidawind, C and Marschalek, R},
title = {Protocol for CRISPR-Cas9-mediated induction of KMT2A rearrangements in cell line and umbilical cord blood hematopoietic stem and progenitor cells.},
journal = {STAR protocols},
volume = {6},
number = {1},
pages = {103481},
pmid = {39700011},
issn = {2666-1667},
mesh = {Humans ; *Myeloid-Lymphoid Leukemia Protein/genetics ; *CRISPR-Cas Systems/genetics ; *Hematopoietic Stem Cells/cytology/metabolism ; *Fetal Blood/cytology ; *Histone-Lysine N-Methyltransferase/genetics ; Gene Editing/methods ; K562 Cells ; Gene Rearrangement/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {KMT2A rearrangements are associated with a poor clinical outcome in infant, pediatric, and adult acute lymphoblastic and myeloid leukemia. Here, we present a protocol to reconstruct chromosomal translocations with different partner genes of KMT2A in vitro. We describe steps for patient-specific single guide RNA (sgRNA) design, optimized sgRNA in vitro transcription, detailed purification of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB), and CRISPR-Cas9 editing of the test cell line K562 as well as UCB HSPCs. The provided methodology is donor independent.},
}

Humans
*Myeloid-Lymphoid Leukemia Protein/genetics
*CRISPR-Cas Systems/genetics
*Hematopoietic Stem Cells/cytology/metabolism
*Fetal Blood/cytology
*Histone-Lysine N-Methyltransferase/genetics
Gene Editing/methods
K562 Cells
Gene Rearrangement/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39673702,
year = {2025},
author = {Zi, H and Peng, X and Du, J and Li, J},
title = {Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.},
journal = {STAR protocols},
volume = {6},
number = {1},
pages = {103490},
pmid = {39673702},
issn = {2666-1667},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Pericytes/cytology/metabolism ; Genes, Reporter/genetics ; Animals, Genetically Modified ; RNA, Guide, CRISPR-Cas Systems/genetics ; Blood-Brain Barrier/metabolism ; Zebrafish Proteins/genetics/metabolism ; Receptor, Platelet-Derived Growth Factor beta/genetics/metabolism ; },
abstract = {Pericytes, the mural cells that envelop small blood vessels, play crucial roles in the formation of the blood-brain barrier (BBB). Here, we present a protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique. We describe steps for identifying efficient single guide RNA (sgRNA), constructing donor plasmid, and generating and maintaining the knockin line. We then detail procedures for in vivo imaging of brain pericytes. This protocol is adaptable for creating other knockin lines for specific cell labeling. For complete details on the use and execution of this protocol, please refer to Zi et al.[1].},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Pericytes/cytology/metabolism
Genes, Reporter/genetics
Animals, Genetically Modified
RNA, Guide, CRISPR-Cas Systems/genetics
Blood-Brain Barrier/metabolism
Zebrafish Proteins/genetics/metabolism
Receptor, Platelet-Derived Growth Factor beta/genetics/metabolism

@article {pmid40121088,
year = {2025},
author = {Williams, AA and Redman, O and Domgaard, H and Armbrust, MJ and Jackson, RN},
title = {Determining the biochemical function of type IV CRISPR ribonucleoprotein complexes and accessory proteins.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {79-114},
doi = {10.1016/bs.mie.2025.01.039},
pmid = {40121088},
issn = {1557-7988},
mesh = {*Ribonucleoproteins/metabolism/genetics ; *Pseudomonas aeruginosa/genetics/metabolism ; Bacterial Proteins/metabolism/genetics ; CRISPR-Cas Systems ; DNA Helicases/metabolism/genetics/chemistry ; Plasmids/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Type IV CRISPR systems are phylogenetically diverse and poorly understood. However, recently, major strides have been made toward understanding type IV-A systems. In type IV-A systems, a multi-subunit ribonucleoprotein complex, called the Csf complex, uses a CRISPR-derived guide to bind double-stranded DNA, forming an R-loop to which a helicase called CRISPR-associated DinG (CasDinG) is recruited. It is proposed that the ATP-dependent helicase activity of CasDinG then unwinds duplex DNA near the targeting site, impairing RNA transcription, and gene expression. Here we describe methods used to investigate the type IV-A system from Pseudomonas aeruginosa strain 83 including a plasmid clearance assay, expression and purification of type IV ribonucleoprotein complexes and proteins, nucleic acid binding assays, and CasDinG helicase assays. These methods provide a foundation for future work aimed at understanding these enigmatic systems.},
}

*Ribonucleoproteins/metabolism/genetics
*Pseudomonas aeruginosa/genetics/metabolism
Bacterial Proteins/metabolism/genetics
CRISPR-Cas Systems
DNA Helicases/metabolism/genetics/chemistry
Plasmids/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40121087,
year = {2025},
author = {Wang, S and Chang, L},
title = {Biochemical reconstitution of a type I-B CRISPR-associated transposon.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {55-79},
doi = {10.1016/bs.mie.2025.01.042},
pmid = {40121087},
issn = {1557-7988},
mesh = {*DNA Transposable Elements ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-associated transposons (CASTs) are potential gene editing tools because of their RNA-guided DNA insertion activity. It is essential to understand the mechanisms underlying the transposition for the application of CASTs. Here, we provide protocols for the biochemical reconstitution of a type I-B CAST for RNA-guided transposition. The procedures may be applicable to other types of CASTs and facilitate the mechanism studies of various CASTs.},
}

*DNA Transposable Elements
*Gene Editing/methods
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40121086,
year = {2025},
author = {Pattali, RK and Ornelas, IJ and Nguyen, CD and Xu, D and Divekar, NS and Nuñez, NK},
title = {CRISPRoff epigenome editing for programmable gene silencing in human cell lines and primary T cells.},
journal = {Methods in enzymology},
volume = {712},
number = {},
pages = {517-551},
doi = {10.1016/bs.mie.2025.01.010},
pmid = {40121086},
issn = {1557-7988},
mesh = {Humans ; *Gene Editing/methods ; *Gene Silencing ; *CRISPR-Cas Systems ; *T-Lymphocytes/metabolism ; DNA Methylation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection/methods ; Epigenome ; Epigenesis, Genetic ; Cell Line ; },
abstract = {The advent of CRISPR-based technologies has enabled the rapid advancement of programmable gene manipulation in cells, tissues, and whole organisms. An emerging platform for targeted gene perturbation is epigenetic editing, the direct editing of chemical modifications on DNA and histones that ultimately results in repression or activation of the targeted gene. In contrast to CRISPR nucleases, epigenetic editors modulate gene expression without inducing DNA breaks or altering the genomic sequence of host cells. Recently, we developed the CRISPRoff epigenetic editing technology that simultaneously establishes DNA methylation and repressive histone modifications at targeted gene promoters. Transient expression of CRISPRoff and the accompanying single guide RNAs in mammalian cells results in transcriptional repression of targeted genes that is memorized heritably by cells through cell division and differentiation. Here, we describe our protocol for the delivery of CRISPRoff through plasmid DNA transfection, as well as the delivery of CRISPRoff mRNA, into transformed human cell lines and primary immune cells. We also provide guidance on evaluating target gene silencing and highlight key considerations when utilizing CRISPRoff for gene perturbations. Our protocols are broadly applicable to other CRISPR-based epigenetic editing technologies, as programmable genome manipulation tools continue to evolve rapidly.},
}

Humans
*Gene Editing/methods
*Gene Silencing
*CRISPR-Cas Systems
*T-Lymphocytes/metabolism
DNA Methylation
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection/methods
Epigenome
Epigenesis, Genetic
Cell Line