@article {pmid40029441,
year = {2025},
author = {Pei, Y and Cao, W and Kong, X and Wang, S and Sun, Z and Zuo, Y and Hu, Z},
title = {CRISPR/Cas9-mediated efficient PlCYP81Q38 mutagenesis in Phryma leptostachya.},
journal = {Planta},
volume = {261},
number = {4},
pages = {73},
pmid = {40029441},
issn = {1432-2048},
support = {2023-JC-QN-0213//Natural Science Foundation of Shaanxi Province/ ; 2023YFD1700700//National Key Research and Development Program of China/ ; 2452020221//Chinese Universities Scientific Fund from Science/ ; },
mesh = {*CRISPR-Cas Systems ; *Lignans/metabolism ; *Gene Editing/methods ; *Mutagenesis ; Plant Proteins/genetics/metabolism ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Dioxoles ; Plant Roots/genetics/metabolism ; },
abstract = {Combined with hairy root transformation, the CRISPR/Cas9 system was established to initiate targeted mutagenesis of PlCYP81Q38, which influenced lignan accumulation in Phryma leptostachya. Phryma leptostachya is a traditional Chinese medicinal herb renowned for its applications in both conventional medicine and natural botanical insecticides, with lignans as the main active ingredients. During the biosynthesis of lignans, PlCYP81Q38, a P450 protein, is assumed to play a crucial role and is accountable for the production of sesamin from (+)-pinoresinol. As a cutting-edge genome editing tool, the CRISPR/Cas9 system is widely employed across diverse species for gene functional research but yet to be harnessed in P. leptostachya. This study utilized the CRISPR/Cas9 system in conjunction with hairy root transformation to initiate targeted mutagenesis in PlCYP81Q38 gene. Employing binary vectors, pYLCRISPR/Cas9Pubi-H, complemented by dual single-stranded guided RNAs (sgRNAs), enabled precise editing at two gene sites and the deletion of large fragments. This editing system resulted in mutagenesis rates surpassing 79%, achieving a notable rate of 61.9% fragment deletion mutants. Liquid chromatography/tandem mass spectrometry confirmed the impact on lignan biosynthesis by PlCYP81Q38-targeted mutagenesis, resulting in the accumulation of pinoresinol and disrupted production of sesamin, 6-demethoxy-leptostachyol acetate, and leptostachyol acetate. Furthermore, the knockout of PlCYP81Q38 up-regulated its upstream pathway genes, such as dirigent gene, cinnamoyl-CoA reductase genes, cinnamyl-alcohol dehydrogenase genes, and p-coumarate 3-hydroxylase genes, identified through gene co-expression analysis. Collectively, mediated by the CRISPR/Cas9 platform, the new biotechnology for targeted genome editing within P. leptostachya, our findings affirm the significant roles of PlCYP81Q38 in the lignan biosynthesis pathway and highlight the potential of CRISPR/Cas9 in exploring the functional genome and secondary metabolite biosynthesis of this plant species.},
}

*CRISPR-Cas Systems
*Lignans/metabolism
*Gene Editing/methods
*Mutagenesis
Plant Proteins/genetics/metabolism
Cytochrome P-450 Enzyme System/genetics/metabolism
Dioxoles
Plant Roots/genetics/metabolism

@article {pmid40029034,
year = {2025},
author = {Cheng, X and Zhao, W and Chen, D and Ren, D and Qian, T and Xia, X and Wang, X and Li, Q and Yang, J and Gu, Y and Zhang, P and Yin, K and Yu, P and Dong, W},
title = {Ultrasensitive Detection of FEN1 Activity for Cancer Diagnosis Using a CRISPR/Cas13a-Based Triple Cascade Amplification System.},
journal = {Advanced healthcare materials},
volume = {14},
number = {6},
pages = {e2404411},
doi = {10.1002/adhm.202404411},
pmid = {40029034},
issn = {2192-2659},
support = {22104090//National Natural Science Foundation of China/ ; 82102184//National Natural Science Foundation of China/ ; 82204349//National Natural Science Foundation of China/ ; 82170526//National Natural Science Foundation of China/ ; 62305207//National Natural Science Foundation of China/ ; PT21011//Shanghai High-Level Local University construction project/ ; 23ZR1436800//Shanghai 2023 "Science and Technology Innovation Action Plan" Natural Science Foundation Project/ ; ZYYZDXK-2023070//NATCM's Project of High-level Construction of Key TCM Disciplines/ ; 2023AH010073//Program for Excellent Sci-tech Innovation Teams of Universities in Anhui Province/ ; LCZX2201//Clinical Research Foundation of Huadong Hospital/ ; //the Junior Thousand Talents Program of China/ ; },
mesh = {Humans ; *Flap Endonucleases/metabolism ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/metabolism/genetics ; Biosensing Techniques/methods ; Cell Line, Tumor ; },
abstract = {Flap endonuclease 1 (FEN1) is closely associated with tumor progression and proliferation, making it a promising biomarker for cancer diagnosis. However, developing a sensitive, reliable, and user-friendly method for quantitative FEN1 detection remains technically challenging. In this study, an ultrasensitive FEN1 biosensor is established using a target-induced cleavage-ligation-transcription-activation cascade strategy (LTACas13a) to enhance the cleavage ability of CRISPR/Cas13a. The LTACas13a method has shown excellent performance in screening FEN1 inhibitors and detecting endogenous FEN1 activity in living cells, as well as in clinical biological samples such as human serum and tissue samples. Additionally, using a universal dumbbell probe derived from FEN1, a multiplex LTACas13a strategy is developed for detecting various DNA glycosylases, including formamidopyrimidine DNA glycosylase, uracil DNA glycosylase, and human alkyl adenine DNA glycosylase. This straightforward approach provides a reliable and effective diagnostic tool for early-stage cancer detection and offers significant opportunities for FEN1 biosensing and related drug discovery.},
}

Humans
*Flap Endonucleases/metabolism
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/metabolism/genetics
Biosensing Techniques/methods
Cell Line, Tumor

@article {pmid40027883,
year = {2025},
author = {Zhang, X and Tao, C and Li, M and Zhang, S and Liang, P and Huang, Y and Liu, H and Wang, Y},
title = {Engineering of SauriCas9 with enhanced specificity.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {1},
pages = {102455},
pmid = {40027883},
issn = {2162-2531},
abstract = {SauriCas9 is a compact Cas9 nuclease showing promise for in vivo therapeutic applications. However, concerns about off-target effects necessitated improvements in specificity. We addressed this by introducing mutations to eliminate polar contacts between Cas9 and the target DNA, resulting in the SauriCas9-R253A variant with enhanced specificity. To validate its efficacy, we employed SauriCas9-R253A to disrupt three genes (B2M, TRAC, and PDCD1), a strategy integral to the development of allogeneic chimeric antigen receptor T cell (CAR-T) therapies. Our results demonstrated that the most efficient single-guide RNAs for SauriCas9-R253A exhibited comparable activity to SpCas9 and showed no detectable off-target effects in the disruption of these genes, highlighting its therapeutic potential.},
}

@article {pmid40026698,
year = {2025},
author = {Álvarez-Rodríguez, A and Li, Z and Jin, BK and Stijlemans, B and Geldhof, P and Magez, S},
title = {A CRISPR-Cas-based recombinase polymerase amplification assay for ultra-sensitive detection of active Trypanosoma brucei evansi infections.},
journal = {Frontiers in molecular biosciences},
volume = {12},
number = {},
pages = {1512970},
pmid = {40026698},
issn = {2296-889X},
abstract = {INTRODUCTION: Control of Trypanosoma brucei evansi (T. b. evansi) infections remains a significant challenge in managing Surra, a widespread veterinary disease affecting both wild and domestic animals. In the absence of an effective vaccine, accurate diagnosis followed by treatment is crucial for successful disease management. However, existing diagnostic methods often fail to detect active infections, particularly in field conditions. Recent advancements in CRISPR-Cas technology, combined with state-of-the-art isothermal amplification assays, offer a promising solution. This approach has led us to the development of a TevRPA-CRISPR assay, a highly sensitive and specific T. b. evansi diagnostic tool suitable for both laboratory and field settings.

METHODS: First, the TevCRISPR-Cas12b cleavage assay was developed and optimized, and its analytical sensitivity was evaluated. Next, this technology was integrated with the TevRPA to create the TevRPA-CRISPR test, with the reaction conditions being optimized and its analytical sensitivity and specificity assessed. Finally, the test's accuracy in detecting both active and cured T. b. evansi infections was evaluated.

RESULTS: The optimized TevCRISPR-Cas12b cleavage assay demonstrated the ability to detect T. b. evansi target DNA at picomolar concentrations. Integrating TevCRISPR-Cas12b with RPA in Two-Pot and One-Pot TevRPA-CRISPR tests achieved up to a 100-fold increase in analytical sensitivity over RPA alone, detecting attomolar concentrations of T. b. evansi target DNA, while maintaining analytical specificity for T. b. evansi. Both assays exhibited performance comparable to the gold standard TevPCR in experimental mouse infections, validating their effectiveness for detecting active infections and assessing treatment efficacy.

DISCUSSION: The TevRPA-CRISPR tests prove highly effective for diagnosing active infections and assessing treatment efficacy, while being adaptable for both laboratory and field use. Thus, the TevRPA-CRISPR assays emerge as a promising addition to current diagnostic tools, offering efficient and reliable detection of active T. b. evansi infections.},
}

@article {pmid40025688,
year = {2025},
author = {He, F and Liu, X and Wang, H and Li, X and Wu, Y and Zhang, D and Liang, S},
title = {The Transcriptional Regulator DhyR Positively Modulates Daptomycin Biosynthesis in Streptomyces roseosporus.},
journal = {Microbial biotechnology},
volume = {18},
number = {3},
pages = {e70110},
doi = {10.1111/1751-7915.70110},
pmid = {40025688},
issn = {1751-7915},
support = {2023SCUH0068//Basic Research Funding for Colleges and Universities- 'From 0 to 1' Innovative Research Program of Sichuan University/ ; 2024ZYD0107//Sichuan Province Science and Technology Support Program/ ; },
mesh = {*Streptomyces/genetics/metabolism ; *Daptomycin/biosynthesis ; *Gene Expression Regulation, Bacterial ; *Multigene Family ; Biosynthetic Pathways/genetics ; Transcription Factors/genetics/metabolism ; Gene Expression Profiling ; Anti-Bacterial Agents/biosynthesis/metabolism ; Gene Deletion ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Daptomycin (DAP) is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, and its biosynthesis is precisely regulated by a complex regulatory network. Although the biosynthetic pathway of DAP has been elucidated, the regulatory mechanism governing its biosynthesis at the transcriptional level is not yet fully understood. In the present study, a new transcriptional regulator, DhyR, was identified. A deletion mutant of dhyR was constructed using the CRISPR-Cas9 tool to elucidate the biological role of DhyR thanks to functional and transcriptomic analyses. The results demonstrated that DhyR positively regulates DAP biosynthesis in S. roseosporus. The in-frame deletion of the dhyR gene resulted in a significant downregulation of the transcription levels of all structural genes within the DAP biosynthetic gene cluster and a significant decrease in DAP yield. In contrast, overexpression of dhyR enhanced the transcription levels of the DAP biosynthetic gene cluster, leading to a 23% increase in DAP yield. Deletion of dhyR caused significant changes in the expression of multiple genes involved in carbohydrate metabolism, energy metabolism and amino acid metabolic pathways through transcriptome analysis. Especially, deletion of dhyR led to a significant downregulation of transcription levels of three DAP biosynthesis-associated genes, including atrA, depR1 and ssig-05090. In summary, DhyR positively regulates DAP biosynthesis in S. roseosporus by influencing the expression of the DAP gene cluster and modulating precursor flux. It functions as a pleiotropic regulator of primary and secondary metabolism in S. roseosporus.},
}

*Streptomyces/genetics/metabolism
*Daptomycin/biosynthesis
*Gene Expression Regulation, Bacterial
*Multigene Family
Biosynthetic Pathways/genetics
Transcription Factors/genetics/metabolism
Gene Expression Profiling
Anti-Bacterial Agents/biosynthesis/metabolism
Gene Deletion
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40024074,
year = {2025},
author = {Mukherjee, AG and R, K and Gopalakrishnan, AV},
title = {Recent advances in CRISPR technology in the milieu of oral squamous cell carcinoma.},
journal = {Pathology, research and practice},
volume = {269},
number = {},
pages = {155860},
doi = {10.1016/j.prp.2025.155860},
pmid = {40024074},
issn = {1618-0631},
abstract = {Oral squamous cell carcinoma (OSCC) is a widely recognized cancer that constitutes over 90 % of all oral cancer cases [1]. Thanks to advancements in cancer treatment, such as radiation, chemotherapy, and molecular target therapy, the 5-year relative survival rate for OSCC has more than doubled in the past 26 years. However, the exact cause and molecular process of OSCC have not been fully understood, and further investigation is necessary. CRISPR/Cas technology is seen as a groundbreaking tool in molecular biology, providing unparalleled accuracy in altering genes. Its use in conditions like OSCC shows excellent potential for advancing research and treatment development. OSCC, characterized by the growth of cancer cells in the lining of the mouth, presents challenges in its treatment and control. Traditional therapies like surgery, radiation, and chemotherapy often show limited success and may lead to severe side effects. The promise of CRISPR/Cas technology in tackling critical aspects of OSCC is apparent. In this correspondence, we discuss the recent advances in CRISPR technology in the OSCC milieu.},
}

@article {pmid40022609,
year = {2025},
author = {Lee, WH and Bates, EA and Kipp, ZA and Pauss, SN and Martinez, GJ and Blair, CA and Hinds, TD},
title = {Insulin receptor responsiveness governs TGFβ-induced hepatic stellate cell activation: Insulin resistance instigates liver fibrosis.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {5},
pages = {e70427},
pmid = {40022609},
issn = {1530-6860},
support = {R01DK121797//HHS | NIH | NIDDK | Division of Diabetes, Endocrinology, and Metabolic Diseases (DEM)/ ; R01DA058933//HHS | NIH | National Institute on Drug Abuse (NIDA)/ ; F31HL170972//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; F31HL175979//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 25PRE1374495//American Heart Association (AHA)/ ; },
mesh = {Cell Line ; Humans ; *Insulin Resistance ; *Receptor, Insulin/metabolism ; *Hepatic Stellate Cells/metabolism ; Transforming Growth Factor beta/metabolism ; CRISPR-Cas Systems ; *Liver Cirrhosis/metabolism ; Cell Proliferation ; Cell Movement ; Signal Transduction ; Gene Regulatory Networks ; },
abstract = {The insulin receptor (INSR) has been shown to be hyperactive in hepatic stellate cells (HSCs) in humans and rodents with liver fibrosis. To explore HSC cellular mechanisms that INSR regulates during pro-fibrotic stimulation, we used CRISPR-Cas9 technology. We knocked out a portion of the INSR gene in human LX2 HSC cells (INSR[e5-8] KO) that regulates insulin responsiveness but not the insulin-like growth factor (IGF) or transforming growth factor-β (TGFβ) signaling. The INSR[e5-8] KO HSCs had significantly higher cell growth, BrdU incorporation, and lower TP53 expression that suppresses growth, and they also exhibited increased migration compared to the Scramble control. We treated the scramble control and INSR[e5-8] KO HSCs with insulin or TGFβ and profiled hundreds of kinase activities using the PamGene PamStation kinome technology. Our analysis showed that serine/threonine kinase (STK) activities were reduced, and most of the protein-tyrosine kinase (PTK) activities were increased in the INSR[e5-8] KO compared to the Scramble control HSCs. To study gene transcripts altered in activated Scramble control and INSR[e5-8] KO HSCs, we treated them with TGFβ for 24 h. We isolated RNA for sequencing and found that the INSR[e5-8] KO cells, compared to control HSCs, had altered transcriptional responsiveness to TGFβ stimulation, collagen-activated signaling, smooth muscle cell differentiation pathways, SMAD protein signaling, collagen metabolic process, integrin-mediated cell adhesion, and notch signaling. This study demonstrates that reduced INSR responsiveness enhances HSC growth and selectively mediates TGFβ-induced HSC activation. These findings provide new insights into the development of more effective treatments for liver fibrosis.},
}

Cell Line
Humans
*Insulin Resistance
*Receptor, Insulin/metabolism
*Hepatic Stellate Cells/metabolism
Transforming Growth Factor beta/metabolism
CRISPR-Cas Systems
*Liver Cirrhosis/metabolism
Cell Proliferation
Cell Movement
Signal Transduction
Gene Regulatory Networks

@article {pmid40021632,
year = {2025},
author = {Gao, S and Weng, B and Wich, D and Power, L and Chen, M and Guan, H and Ye, Z and Xu, C and Xu, Q},
title = {Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2081},
pmid = {40021632},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *Adenine/metabolism/chemistry ; Humans ; Streptococcus pyogenes/genetics/enzymology ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Protein Domains ; HEK293 Cells ; Bacterial Proteins/genetics/metabolism/chemistry ; },
abstract = {Domain expansion contributes to diversification of RNA-guided-endonucleases including Cas9. However, it remains unclear how REC domain expansion could benefit Cas9. In this study, we identify an insertion spot that is compatible with large REC insertion and succeeds in enlarging the non-catalytic REC domain of Streptococcus pyogenes Cas9. The natural-evolution-like giant SpCas9 (GS-Cas9) is created and shows substantially improved editing precision. We further discover that enlarging the REC domain could enable regulation of the N-terminal adenine deaminase TadA8e tethered to the Cas9 scaffold, which contributes to substantially reducing unexpected editing and improving the precision of the adenine base editor ABE8e. We provide proof of concept for evolution-inspired expansion of Cas9 and offer an alternative solution for optimizing gene editors. Our study also indicates a vast potential for engineering the topological malleability of RNA-guided endonucleases and base editors.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
*Adenine/metabolism/chemistry
Humans
Streptococcus pyogenes/genetics/enzymology
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Protein Domains
HEK293 Cells
Bacterial Proteins/genetics/metabolism/chemistry

@article {pmid40021494,
year = {2025},
author = {Sajjad, MW and Imran, I and Muzamil, F and Naqvi, RZ and Amin, I},
title = {AZD7648 (DNA-PKcs inhibitor): a two-edged sword for editing genomes.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {49},
pmid = {40021494},
issn = {1438-7948},
mesh = {*Gene Editing ; *DNA-Activated Protein Kinase/genetics/antagonists & inhibitors ; Humans ; CRISPR-Cas Systems ; DNA End-Joining Repair/drug effects ; Recombinational DNA Repair ; Animals ; Pyrimidines ; Schiff Bases ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) has been the most practical technique in genome editing for the last decade. Its molecular mechanism includes steps that occur in a sequence, starting from a break in a double strand to repair. After a double-strand break in the DNA strand, the repairing of DNA done via Homology-Directed Repair (HDR) is considered important in different organisms as it is ideal for precise genome editing and the reduction of unintended mutations. Still, it is mostly dominated by the Non-Homologous End Joining (NHEJ) pathway. A recent study by Cullot et al. published in Nature Biotechnology showed interesting features of AZD7648 (a DNA-PKcs inhibitor) that increase the probability of HDR event while DNA repairing (Cullot et al. 2024).},
}

*Gene Editing
*DNA-Activated Protein Kinase/genetics/antagonists & inhibitors
Humans
CRISPR-Cas Systems
DNA End-Joining Repair/drug effects
Recombinational DNA Repair
Animals
Pyrimidines
Schiff Bases

@article {pmid40020637,
year = {2025},
author = {Deng, A and Mao, Z and Jin, X and Lv, W and Huang, L and Zhong, H and Wang, S and Shi, Y and Zhou, T and Zhao, J and Huang, Q and Luo, X and Ma, L and Zou, H and Fu, R and Huang, G},
title = {ID-CRISPR: A CRISPR/Cas12a platform for label-free and sensitive detection of rare mutant alleles using self-interference DNA hydrogel reporter.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117309},
doi = {10.1016/j.bios.2025.117309},
pmid = {40020637},
issn = {1873-4235},
abstract = {Accurate and sensitive detection of single nucleotide variants (SNVs) is paramount for cancer diagnosis and treatment. The CRISPR/Cas12a system shows promise for SNV detection due to its high sensitivity and single-base specificity. However, most CRISPR/Cas12a-based methods rely on F/Q-labeled single-stranded DNA (ssDNA) reporters, which are susceptible to fluorescence fluctuations, thereby reducing accuracy. To address these limitations, researchers have proposed using DNA hydrogels as signal transducers in CRISPR/Cas12a systems. Yet, the encapsulation of indicators into DNA hydrogels introduces additional instability, which could compromise both detection sensitivity and linearity. In this study, we integrated hyperspectral interferometry into a DNA hydrogel-based CRISPR/Cas12a detection platform (ID-CRISPR) to achieve sensitive label-free SNV detection. Using EGFR L858R SNV as a model target, we demonstrated that ID-CRISPR can detect mutant allele frequencies (MAFs) as low as 0.1% with a limit of detection (LOD) of 5 aM, while also showing its potential for quantifying SNV abundance. Its clinical utility was confirmed through analysis of lung tumor samples, with results consistent with sequencing data. Therefore, ID-CRISPR provides a sensitive, label-free, and user-friendly platform for SNV detection, offering new insights into combining optical sensing with DNA hydrogel technology in CRISPR/Cas assays.},
}

@article {pmid39970722,
year = {2025},
author = {Lai, Y and Wang, J and Xie, N and Liu, G and Lacap-Bugler, DC},
title = {Identification of a novel forkhead transcription factor MtFKH1 for cellulase and xylanase gene expression in Myceliophthora thermophila (ATCC 42464).},
journal = {Microbiological research},
volume = {294},
number = {},
pages = {128097},
doi = {10.1016/j.micres.2025.128097},
pmid = {39970722},
issn = {1618-0623},
mesh = {*Gene Expression Regulation, Fungal ; *Cellulase/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; *Sordariales/genetics/enzymology ; *Forkhead Transcription Factors/genetics/metabolism ; *Promoter Regions, Genetic ; Endo-1,4-beta Xylanases/genetics/metabolism ; Cellulose/metabolism ; Gene Expression Profiling ; CRISPR-Cas Systems ; Spores, Fungal/genetics ; },
abstract = {Myceliophthora thermophila is a thermophilic fungus, known to produce industrially important enzymes in biorefineries. The mechanism underlying cellulase and xylanase expression in filamentous fungi is a complex regulatory network controlled by numerous transcription factors (TFs). These TFs in M. thermophila remain unclear. Here, we identified and characterised a novel cellulase and xylanase regulator MtFKH1 in M. thermophila through comparative transcriptomic and genetic analyses. Five of the eight potential TFs, which showed differential expression levels when grown on Avicel and glucose, were successfully deleted using the newly designed CRISPR/Cas9 system. This system identified the forkhead TF MtFKH1. The disruption of Mtfkh1 elevated the cellulolytic and xylanolytic enzyme activities, whereas the overexpression of Mtfkh1 led to considerable decrease in cellulase and xylanase production in M. thermophila cultivated on Avicel. The loss of Mtfkh1 also exhibited an impairment in sporulation in M. thermophila. Real-time quantitative reverse transcription PCR (RT-qPCR) and the electrophoretic mobility shift assays (EMSAs) demonstrated that MtFKH1 regulates the gene expression and specifically bind to the promoter regions of genes encoding β-glucosidase (bgl1/MYCTH_66804), cellobiohydrolase (cbh1/MYCTH_109566), and xylanase (xyn1/MYCTH_112050), respectively. Furthermore, DNase I footprinting analysis identified binding motif of MtFKH1 in the upstream region of Mtbgl1, with strongest binding affinity. Finally, transcriptomic profiling and Gene Ontology (GO) enrichment analyses of Mtfkh1 deletion mutant revealed that the regulon of MtFKH1 were significantly prevalent in hydrolase activity (acting on glycosyl bonds), polysaccharide binding, and carbohydrate metabolic process functional categories. These findings expand our knowledge on how forkhead transcription factor regulates lignocellulose degradation and provide a novel target for engineering of fungal cell factories with the hyperproduction of cellulase and xylanase.},
}

*Gene Expression Regulation, Fungal
*Cellulase/genetics/metabolism
*Fungal Proteins/genetics/metabolism
*Sordariales/genetics/enzymology
*Forkhead Transcription Factors/genetics/metabolism
*Promoter Regions, Genetic
Endo-1,4-beta Xylanases/genetics/metabolism
Cellulose/metabolism
Gene Expression Profiling
CRISPR-Cas Systems
Spores, Fungal/genetics

@article {pmid39956401,
year = {2025},
author = {Puri, B and Majumder, S and Gaikwad, AB},
title = {CRISPR/Cas9 based knockout of lncRNA MALAT1 attenuates TGF-β1 induced Smad 2/3 mediated fibrosis during AKI-to-CKD transition.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {207},
number = {},
pages = {107044},
doi = {10.1016/j.ejps.2025.107044},
pmid = {39956401},
issn = {1879-0720},
mesh = {*RNA, Long Noncoding/genetics ; Animals ; *Acute Kidney Injury/genetics/metabolism ; *Fibrosis/genetics ; *Smad3 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Rats ; *Transforming Growth Factor beta1/genetics/metabolism ; Cell Line ; *Renal Insufficiency, Chronic/genetics/metabolism ; *Smad2 Protein/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Acute kidney injury (AKI) is a significant clinical issue with potential long-term consequences, as even a single episode can progress to chronic kidney disease (CKD). The AKI-to-CKD transition involves complex pathophysiology, including persistent inflammation, apoptosis, and fibrosis. Long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been recognized as a potential therapeutic target for various kidney diseases, including AKI and CKD. In our previous study, we conducted the transcriptomic analysis of lncRNAs in-vitro and animal models of AKI-to-CKD transition and found several dysregulated lncRNAs such as MALAT1, MEG3, NEAT1, MIAT, and H19 in this transition. Among these, we have selected lncRNA MALAT1 to further validate its role in AKI-to-CKD transition as a therapeutic target via a cluster regularly intercept short palindromic protein (CRISPR) associated protein 9 (Cas9)-mediated knockout approach in NRK52E cells. Guide RNAs (gRNAs) were designed to target MALAT1, and the PX459 turbo green fluorescence protein (GFP) plasmid containing MALAT1 gRNA1&2 was transfected into NRK52E cells using CRISPRMAX. Results demonstrated that MALAT1 knockout significantly reduced MALAT1 expression and attenuated Smad2/3-mediated fibrosis by decreasing pSmad2, pSmad2/3, Smad4, vimentin, fibronectin, collagen-I, and α-SMA expression levels, while increasing Smad7, Smurf2, and E-cadherin levels. These findings suggest that targeting the MALAT1/Smad2/3 pathway could be a potential therapeutic target for mitigating fibrosis to prevent AKI-to-CKD transition.},
}

*RNA, Long Noncoding/genetics
Animals
*Acute Kidney Injury/genetics/metabolism
*Fibrosis/genetics
*Smad3 Protein/genetics/metabolism
*CRISPR-Cas Systems
Rats
*Transforming Growth Factor beta1/genetics/metabolism
Cell Line
*Renal Insufficiency, Chronic/genetics/metabolism
*Smad2 Protein/genetics/metabolism
Gene Knockout Techniques

@article {pmid39904299,
year = {2025},
author = {Zhang, Y and Gao, L and Shi, Z and Wu, Q and Miao, X},
title = {Paper-based electrochemiluminescence telomerase activity detection using hybridization chain reaction and CRISPR/Cas12a dual signal amplification.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {164},
number = {},
pages = {108916},
doi = {10.1016/j.bioelechem.2025.108916},
pmid = {39904299},
issn = {1878-562X},
mesh = {*Telomerase/genetics ; Humans ; *Luminescent Measurements/methods ; *Nucleic Acid Hybridization ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Paper ; Biosensing Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Sensitive telomerase activity detection becomes particularly significance since the important value of it in early cancer diagnosis as a potential biomarker. Herein, we developed a paper-based analytical devices (PADs) for telomerase activity detection, using positively charged Au@luminol nanoparticles ((+)Au@luminol NPs) as electrochemiluminescence (ECL) signal probe coupling with hybridization chain reaction (HCR) and CRISPR/Cas12a dual signal amplification. Firstly, the initial strong ECL signal was obtained based on the electrostatic adsorption of (+)Au@luminol NPs onto the surface of HCR double-stranded hybrid aggregates. In the presence of telomerase, the primer was efficiently elongated with telomeric repeats of (TTAGGG)n to release activator DNA and trigger the CRISPR/Cas12a, which can prevent the happen of HCR and the adsorption of (+)Au@luminol NPs through cleaving the capture probe on the electrode surface, such results directly inducing the decrease of the ECL signal that was proportional to telomerase concentration, due to the efficient signal amplification of HCR and CRISPR/Cas12a, a low detection limit of 2.3 cells/mL for telomerase could be detected. Moreover, the sensor realized the effective application for telomerase extracts analysis in human serum samples, making it possess potential application value for telomerase activity assays in cancer diagnostics.},
}

*Telomerase/genetics
Humans
*Luminescent Measurements/methods
*Nucleic Acid Hybridization
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
Paper
Biosensing Techniques/methods
Limit of Detection
Metal Nanoparticles/chemistry
Gold/chemistry
Nucleic Acid Amplification Techniques/methods

@article {pmid39880143,
year = {2025},
author = {Li, L and Luo, M and Zhou, L and Wang, Y and Jiao, Y and Wang, C and Gong, C and Cen, X and Yao, S},
title = {Glucocorticoid pre-administration improves LNP-mRNA mediated protein replacement and genome editing therapies.},
journal = {International journal of pharmaceutics},
volume = {672},
number = {},
pages = {125282},
doi = {10.1016/j.ijpharm.2025.125282},
pmid = {39880143},
issn = {1873-3476},
mesh = {Animals ; *Gene Editing/methods ; *RNA, Messenger/administration & dosage ; *Glucocorticoids/administration & dosage ; *Nanoparticles/administration & dosage ; Mice ; Lipids/chemistry ; Male ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Hepatocytes/metabolism ; Phagocytosis/drug effects ; CRISPR-Cas Systems ; Humans ; Liver/metabolism ; Macrophages/metabolism/drug effects ; Gene Transfer Techniques ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are among the most promising non-viral mRNA delivery systems for gene therapeutic applications. However, the in vivo delivery of LNP-mRNA remains challenging due to multiple intrinsic barriers that hinder LNPs from reaching their target cells. In this study, we sought to enhance LNP delivery by manipulating intrinsic regulatory mechanisms involved in their metabolism. We demonstrated that activation of the glucocorticoid pathway significantly increased the systemic delivery of LNP-mRNA in both mice and monkeys, achieving up to a fourfold improvement. This enhancement was primarily attributed to the glucocorticoid-mediated inhibition of macrophage phagocytosis in circulation and the liver, which resulted in higher LNP accumulation in hepatocytes. Consequently, glucocorticoid activation improved the therapeutic efficacy of LNP-based protein replacement and CRISPR/Cas9 genome editing therapies. Together, these findings establish a practical strategy to enhance the systemic delivery of RNA-based protein replacement and genome editing therapeutics, highlighting the potential of manipulating endogenous mechanisms to optimize exogenous gene delivery.},
}

Animals
*Gene Editing/methods
*RNA, Messenger/administration & dosage
*Glucocorticoids/administration & dosage
*Nanoparticles/administration & dosage
Mice
Lipids/chemistry
Male
Mice, Inbred C57BL
Genetic Therapy/methods
Hepatocytes/metabolism
Phagocytosis/drug effects
CRISPR-Cas Systems
Humans
Liver/metabolism
Macrophages/metabolism/drug effects
Gene Transfer Techniques
Liposomes

@article {pmid39739998,
year = {2025},
author = {Liang, H and Mu, X and Huang, Y and Zhao, S and Tian, J},
title = {Magnetic Assisted DNA Logic Gate Nanomachine Based on CRISPR/Cas12a for Recognition of Dual miRNAs.},
journal = {Chemistry, an Asian journal},
volume = {20},
number = {5},
pages = {e202401209},
doi = {10.1002/asia.202401209},
pmid = {39739998},
issn = {1861-471X},
support = {22064003//National Natural Science Foundation of China/ ; 21465007//National Natural Science Foundation of China/ ; 2015GXNSFGA139003//Guangxi Natural Science Foundation of China/ ; //Bagui Scholar of Guangxi Province/ ; //Duxiu Scholar of Guangxi Normal University/ ; CMEMR2014-A08//State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University/ ; },
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry ; DNA, Catalytic/chemistry/metabolism ; Biosensing Techniques ; Logic ; Computers, Molecular ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The anomalous expression of microRNA poses a serious threat to human life and health safety, and serves as an important biomarker for cancer detection. In this study, a novel magnetic-assisted DNA logic gate nanomachine triggered by miRNA-21 and miRNA-155 was designed based on the trans-cleavage activity of CRISPR/Cas12a activated by a split DNA activator, using only a single crRNA and signal probe, which simplified the detection procedure and complex nucleic acid amplification. The presence of target molecules, miRNA-21 and miRNA-155, can stimulate the DNA walker machine assembled on magnetic beads, which releases activator under the action of DNAzyme. Then the trans-cleavage activity of CRISPR/Cas12a is initiated and the system signal significantly increases. Based on this, an AND logic gate nanomachine was constructed for simultaneous analysis of miRNA-21 and miRNA-155. The detection limits of miRNA-21 and miRNA-15 were 9.00 pM and 42.00 pM, respectively, and this method was successfully applied to miRNA analysis in cell samples. This nanomachine combined the DNA walker with DNA logic circuit and CRISPR/Cas12a system, providing a new approach for simultaneous detection of multiple targets and further expanding the application of gene editing in the analysis and sensing of multiple target substances.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry
DNA, Catalytic/chemistry/metabolism
Biosensing Techniques
Logic
Computers, Molecular
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid38631265,
year = {2024},
author = {Wang, H and Hang, X and Wang, H and Peng, J and Yu, H and Wang, L},
title = {Label/immobilization-free Cas12a-based electrochemiluminescence biosensor for sensitive DNA detection.},
journal = {Talanta},
volume = {275},
number = {},
pages = {126114},
doi = {10.1016/j.talanta.2024.126114},
pmid = {38631265},
issn = {1873-3573},
mesh = {*Luminescent Measurements/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Cas Systems ; Bacterial Proteins/genetics/metabolism ; Feasibility Studies ; Humans ; *DNA, Viral/analysis/genetics ; *Human papillomavirus 16/genetics/isolation & purification ; Hydrogels ; Reproducibility of Results ; *Electrochemical Techniques/methods ; },
abstract = {Electrochemiluminescence (ECL) is one of the most sensitive techniques in the field of diagnostics. However, they typically require luminescent labeling and electrode surface biological modification, which is a time-consuming and laborious process involving multiple steps and may also lead to low reaction efficiency. Fabricating label/modification-free biosensors has become one of the most attractive parts for simplifying the ECL assays. In this work, the ECL luminophores carbon dots (CDs) were encapsulated in DNA hydrogel in situ by a simple rolling circle amplification (RCA) reaction. Upon binding of the target DNA, active Cas12a induces a collateral cleavage of the hydrogel's ssDNA backbone, resulting in a programmable degradation of the hydrogel and the release of CDs. By directly measuring the released CDs ECL, a simple and rapid label/modification-free detection of the target HPV-16 was realized. It is noted that this method allowed for 0.63 pM HPV-16 DNA detection without any amplification step, and it could take only ∼60 min for a fast test of a human serum sample. These results showed that our label/modification-free ECL biosensor has great potential for use in simple, rapid, and sensitive point-of-care (POC) detection.},
}

*Luminescent Measurements/methods
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases/genetics/metabolism
CRISPR-Cas Systems
Bacterial Proteins/genetics/metabolism
Feasibility Studies
Humans
*DNA, Viral/analysis/genetics
*Human papillomavirus 16/genetics/isolation & purification
Hydrogels
Reproducibility of Results
*Electrochemical Techniques/methods

@article {pmid40019808,
year = {2025},
author = {Cook, AL and Moyer, AL and Boxer, L and Norris, AL},
title = {Re: Novel Off-Targeting Events Identified after Genome-Wide Analysis of CRISPR-Cas Edited Pigs.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0091},
pmid = {40019808},
issn = {2573-1602},
}

@article {pmid40019770,
year = {2025},
author = {Mao, X and Lu, Y and Gao, Z and Zhong, J and Xiao, A and Lin, J and Hu, J and Shu, B and Zhang, C},
title = {Modular Microfluidic Sensor Integrating Nucleic Acid Extraction, CRISPR/Cas13a, and Electrochemiluminescence for Multichannel RNA Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c06197},
pmid = {40019770},
issn = {1520-6882},
abstract = {Rapid and accurate screening of pathogens is crucial for disease detection. Here, a modular microfluidic sensor has been constructed for RNA detection, with integrated nucleic acid extraction, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a reaction, and electrochemiluminescence (ECL) detection. The sensor consists of nucleic acid processing and detection modules. The nucleic acid processing module is used for nucleic acid extraction, RNA distribution, and the CRISPR/Cas reaction. Specifically, immiscible filtration assisted by surface tension is employed for nucleic acid extraction, significantly reducing the extraction time. Magnetic force is utilized for RNA distribution and transportation, minimizing the need for microstructures, such as microvalves and micropumps. Multichannel CRISPR/Cas13a reactions enable biological recognition, signal amplification, and multiplex detection. The fiber material-based detection module controls fluid flow and performs dry chemistry-based ECL detection. A novel multichannel closed bipolar electrode-based ECL (MCBPE-ECL) system is employed, with simplified experimental operations and enhanced sensitivity. Together, the multichannel CRISPR/Cas13a reactions and MCBPE-ECL enable the sensor's multiplexed detection. Under optimized conditions, the sensor can complete RNA extraction and detection in 30 min, with a detection limit of 0.372 fM for Escherichia coli 16S rRNA. Furthermore, in human blood samples, the detection limit for E. coli is 63.8 cfu/mL. Notably, the sensor can simultaneously determine the growth curves of single colonies of E. coli and Staphylococcus aureus strains in the same culture medium, demonstrating its multiplexed detection capability.},
}

@article {pmid40017424,
year = {2025},
author = {Song, Y and Park, H and Thirumalaraju, P and Kovilakath, N and Hardie, JM and Bigdeli, A and Bai, Y and Chang, S and Yoo, J and Kanakasabapathy, MK and Kim, S and Chun, J and Chen, H and Li, JZ and Tsibris, AM and Kuritzkes, DR and Shafiee, H},
title = {Deactivated Cas9-Engineered Magnetic Micromotors toward a Point-of-Care Digital Viral RNA Assay.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.4c14913},
pmid = {40017424},
issn = {1936-086X},
abstract = {Digital nucleic acid assays, known for their high sensitivity and specificity, typically rely on fluorescent readouts and expensive and complex nanowell manufacturing, which constrain their broader use in point-of-care (POC) application. Here, we introduce an alternative digital molecular diagnostics, termed dCRISTOR, by seamlessly integrating deactivated Cas9 (dCas9)-engineered micromotors, extraction-free loop-mediated isothermal amplification (LAMP), low-cost bright field microscopy, and deep learning-enabled image processing. The micromotor, composed of a polystyrene sphere attached to a magnetic bead, incorporates a dCas9 ribonucleoprotein complex. The presence of human immunodeficiency virus-1 (HIV-1) RNA in a sample results in the formation of large-sized amplicons that can be specifically captured by the micromotors, reducing their velocity induced by an external magnetic field. The micromotor is propelled by an external magnetic field, which eliminates the need for chemical fuels, reducing system complexity, and allowing for precise control over micromotor movement, enhancing accuracy and reliability. A convolutional neural network classification-based multiobject tracking algorithm, CNN-MOT, accurately measures the change in micromotor motion, facilitating the binary digital assay format ("1" or "0") for simplified result interpretation without user bias. Incorporating an extraction-free LAMP assay streamlines the dCRISTOR workflow, enabling qualitative HIV-1 detection in spiked plasma (n = 21) that demonstrates 100% sensitivity and specificity and achieves a limit of detection (LOD) of 0.96 copies/μL. The assay also achieved 100% correlation with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in clinical patient samples (n = 9). The dCRISTOR assay, a label-free digital nucleic acid testing system that eliminates the need for fluorescence readouts, absorbance measurements, or expensive manufacturing processes, represents a substantial advancement in digital viral RNA diagnostics.},
}

@article {pmid40017406,
year = {2025},
author = {Liu, G},
title = {Advancing CRISPR/Cas Biosensing with Integrated Devices.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {575-576},
doi = {10.1021/acssensors.5c00330},
pmid = {40017406},
issn = {2379-3694},
}

@article {pmid39765088,
year = {2025},
author = {Yang, R and Zhao, L and Fang, M and Kong, W and Luan, Y},
title = {CRISPR-Cas12a-driven aptasensor for sensitive detection of alternariol by using a personal glucose meter.},
journal = {Talanta},
volume = {286},
number = {},
pages = {127496},
doi = {10.1016/j.talanta.2024.127496},
pmid = {39765088},
issn = {1873-3573},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Lactones/chemistry ; Limit of Detection ; CRISPR-Associated Proteins/chemistry/genetics ; Humans ; Bacterial Proteins/genetics/chemistry ; Blood Glucose Self-Monitoring ; beta-Fructofuranosidase/chemistry ; Endodeoxyribonucleases ; },
abstract = {Alternariol (AOH) has attracted much attention as an emerging toxin in edible herbs that can pose potential carcinogenic risks to human. However, the rapid detection of AOH to ensure food safety remains a challenge. Here, a CRISPR-Cas12a-mediated aptamer-based sensor (aptasensor) was proposed for the sensitive quantification of AOH by using a personal glucose meter. First, fluorescent probes were used to validate crRNA sequences that could activate Cas12a trans-cleavage activity. Owing to the high affinity of aptamer, different concentrations of AOH were able to release different levels of Cas12a cleavage activity to cut magnetic bead-modified invertase probes. The free invertase was collected to catalyze hydrolysis of sucrose to glucose. After optimizing a series of key parameters, the constructed aptasensor with dual signal amplification of Cas12a and invertase allowed for highly-sensitive AOH detection in a linear range of 0.45-3000 ng/mL with a detection limit of 0.048 ng/mL. In addition, this aptasensor was successfully applied in edible herb samples with satisfactory recovery rates of 92.19-102.40 %. This CRISPR-Cas12a-mediated aptasensing platform provides a simple and powerful alternative for rapid quantitative detection of AOH, which is of great significance for food safety.},
}

*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Lactones/chemistry
Limit of Detection
CRISPR-Associated Proteins/chemistry/genetics
Humans
Bacterial Proteins/genetics/chemistry
Blood Glucose Self-Monitoring
beta-Fructofuranosidase/chemistry
Endodeoxyribonucleases

@article {pmid39724854,
year = {2025},
author = {Li, X and Dong, J and Deng, L and Huo, D and Yang, M and Hou, C},
title = {CRISPR/Cas12a regulated preassembled bulb-shaped G-quadruplex signal unit for FL/CM dual-mode ultrasensitive detection of miRNA-155.},
journal = {Talanta},
volume = {286},
number = {},
pages = {127413},
doi = {10.1016/j.talanta.2024.127413},
pmid = {39724854},
issn = {1873-3573},
mesh = {*G-Quadruplexes ; *MicroRNAs/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; Biosensing Techniques/methods ; Limit of Detection ; Colorimetry/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {High sensitivity and specificity in microRNA detection are of great significance for early cancer screening. This study employed a pre-assembled bulb-shaped G-quadruplex signal unit (G4MB) as a novel and efficient label-free probe. The products amplified by the miRNA-155-targeted exponential amplification reaction (EXPAR) activated the trans-cleavage activity of CRISPR/Cas12a, disrupting the G4MB structure to achieve dual-channel fluorescence/colorimetric (FL/CM) inverse signal output. Due to the strong signal amplification of EXPAR, the highly efficient cleavage by CRISPR/Cas12a, and the ultra-high response signal of the structurally stable G4MB probe, the FL mode achieved a high signal-to-noise ratio (S/N) of approximately 12.5. The CM mode, combined with smart devices for RGB curve adjustment, successfully corrected the background and provided precise and objective image data support while allowing results to be observed with the naked eye. Additionally, the sensor system exhibited high accuracy in complex human serum environments and RNA extracted from three different types of cells. Moreover, the G4MB probe required no complicated labeling, demonstrated structural stability, and had a rapid response. Most importantly, this study analyzed the advantages of the G4MB and applied it to miRNA detection for the first time, providing practical insights for biosensor construction, molecular diagnostics, and clinical applications.},
}

*G-Quadruplexes
*MicroRNAs/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
Biosensing Techniques/methods
Limit of Detection
Colorimetry/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid38145395,
year = {2025},
author = {Mutale-Joan, C and El Arroussi, H},
title = {Biotechnological strategies overcoming limitations to H. pluvialis-derived astaxanthin production and Morocco's potential.},
journal = {Critical reviews in food science and nutrition},
volume = {65},
number = {8},
pages = {1404-1419},
doi = {10.1080/10408398.2023.2294163},
pmid = {38145395},
issn = {1549-7852},
mesh = {*Xanthophylls/metabolism ; *Biotechnology/methods ; Morocco ; *Microalgae/metabolism ; *Biomass ; CRISPR-Cas Systems ; Chlorophyceae/metabolism ; },
abstract = {Haematococcus pluvialis is the richest source of natural astaxanthin, but the production of H. pluvialis-derived astaxanthin is usually limited by its slow cell proliferation and astaxanthin accumulation. Efforts to enhance biomass productivity, astaxanthin accumulation, and extraction are ongoing. This review highlights different approaches that have previously been studied in microalgal species for enhanced biomass productivity, as well as optimized methods for astaxanthin accumulation and extraction, and how these methods could be combined to bypass the challenges limiting natural astaxanthin production, particularly in H. pluvialis, at all stages (biomass production, and astaxanthin accumulation and extraction). Biotechnological approaches, such as overexpressing low CO2 inducible genes, utilizing complementary carbon sources, CRISPR-Cas9 bioengineering, and the use of active compounds, for biomass productivity are outlined. Direct astaxanthin extraction from H. pluvialis zoospores and Morocco's potential for microalgal-based astaxanthin production are equally discussed. This review emphasizes the need to engineer an optimized H. pluvialis-derived astaxanthin production system combining two or more of these strategies for increased growth, and astaxanthin productivity, to compete in the larger, lower-priced market in aquaculture and nutraceutical sectors.},
}

*Xanthophylls/metabolism
*Biotechnology/methods
Morocco
*Microalgae/metabolism
*Biomass
CRISPR-Cas Systems
Chlorophyceae/metabolism

@article {pmid40015840,
year = {2025},
author = {Chen, S and Ruan, Y and Li, Z and Zhou, C and Chen, Q and Zhou, X and Zhang, Y and Yang, C and Pan, H},
title = {CRISPR/Cas9-mediated editing of the melanization gene ebony in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.},
journal = {Pesticide biochemistry and physiology},
volume = {208},
number = {},
pages = {106231},
doi = {10.1016/j.pestbp.2024.106231},
pmid = {40015840},
issn = {1095-9939},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Melanins/metabolism ; Insect Proteins/genetics/metabolism ; Lepidoptera/genetics ; RNA Interference ; Pigmentation/genetics ; },
abstract = {The melanization process, which is essential for the proper functioning of the cuticle, has been extensively investigated for its enzymatic roles and physiological effects. Henosepilachna vigintioctopunctata, a significant pest species, presents considerable economic threats. However, due to the variable efficiency of RNA interference for genetic manipulation, establishing a CRISPR/Cas9 system is crucial for providing a more precise and reliable method for functional genomics in this non-model insect. In this study, we first utilized RNAi to investigate Hvebony, which encodes N-β-alanyldopamine, a critical compound in cuticle melanization. Subsequently, we introduced CRISPR/Cas9 for the first time in H. vigintioctopunctata. RNAi experiments revealed that knockdown of Hvebony resulted in abnormal melanin accumulation and low mortality rates, indicating its involvement in cuticle tanning. A novel CRISPR/Cas9 workflow was established, successfully resulting in the knocking out of Hvebony and the creation of a stable mutant strain characterized by dark pigmentation and low fitness costs. This study establishes Hvebony as a promising molecular marker for genetic studies in H. vigintioctopunctata. Moreover, it can be utilized in the development of genome editing control strategies and for analyses of gene function in H. vigintioctopunctata.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Melanins/metabolism
Insect Proteins/genetics/metabolism
Lepidoptera/genetics
RNA Interference
Pigmentation/genetics

@article {pmid40015249,
year = {2025},
author = {Dederer, HG},
title = {Human health and genetic technology.},
journal = {Trends in biotechnology},
volume = {43},
number = {3},
pages = {522-532},
doi = {10.1016/j.tibtech.2024.12.006},
pmid = {40015249},
issn = {1879-3096},
mesh = {Humans ; *Genetic Therapy/methods ; Organisms, Genetically Modified/genetics ; Animals ; DNA, Recombinant/genetics ; },
abstract = {The 1975 Asilomar conference contributed to the misperception that recombinant DNA (rDNA) technology is inherently risky to human health and the environment. It thus paved the way toward process-based regulation of genetically modified organisms (GMOs), such as in the EU. Initially, this regulatory approach obstructed technological uses of rDNA related to human health. However, regulators gradually softened the rules applicable to laboratories or industrial facilities. This encouraged R&D and production of pharmaceuticals derived from GMOs. Nevertheless, administering pharmaceuticals containing GMOs to patients may still be confronted with burdensome process-based GMO law on the deliberate release of GMOs into the environment. On the other hand, pharmaceutical law may need to be updated regarding, for example, novel gene therapies or xenotransplantation.},
}

Humans
*Genetic Therapy/methods
Organisms, Genetically Modified/genetics
Animals
DNA, Recombinant/genetics

@article {pmid40014250,
year = {2025},
author = {Bolideei, M and Barzigar, R and Gahrouei, RB and Mohebbi, E and Haider, KH and Paul, S and Paul, MK and Mehran, MJ},
title = {Applications of Gene Editing and Nanotechnology in Stem Cell-Based Therapies for Human Diseases.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {40014250},
issn = {2629-3277},
abstract = {Stem cell research is a dynamic and fast-advancing discipline with great promise for the treatment of diverse human disorders. The incorporation of gene editing technologies, including ZFNs, TALENs, and the CRISPR/Cas system, in conjunction with progress in nanotechnology, is fundamentally transforming stem cell therapy and research. These innovations not only provide a glimmer of optimism for patients and healthcare practitioners but also possess the capacity to radically reshape medical treatment paradigms. Gene editing and nanotechnology synergistically enhance stem cell-based therapies' precision, efficiency, and applicability, offering transformative potential for treating complex diseases and advancing regenerative medicine. Nevertheless, it is important to acknowledge that these technologies also give rise to ethical considerations and possible hazards, such as inadvertent genetic modifications and the development of genetically modified organisms, therefore creating a new age of designer infants. This review emphasizes the crucial significance of gene editing technologies and nanotechnology in the progress of stem cell treatments, particularly for degenerative pathologies and injuries. It emphasizes their capacity to restructure and comprehensively revolutionize medical treatment paradigms, providing fresh hope and optimism for patients and healthcare practitioners.},
}

@article {pmid40014102,
year = {2025},
author = {Jiang, Y and Xue, R and Chang, Y and Cao, D and Liu, B and Li, Y},
title = {The knockout of Gγ subunit HvGS3 by CRISPR/Cas9 gene editing improves the lodging resistance of barley through dwarfing and stem strengthening.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {3},
pages = {61},
pmid = {40014102},
issn = {1432-2242},
support = {2023-NK-A3//Geomatics Technology and Application Key Laboratory of Qinghai Province/ ; 077GJHZ2023028GC//Global Common Challenges Project of International Partnership Program of Chinese Academy of Sciences/ ; 2023-1-1//Qinghai Science and Technology Department/ ; },
mesh = {*Hordeum/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing ; *Gibberellins/metabolism ; *Plant Proteins/genetics/metabolism ; *Plant Stems/genetics/growth & development ; *Lignin/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Phenotype ; Gene Knockout Techniques ; },
abstract = {Gγ subunits participate in multiple biological processes, but their biological function in barley is unknown. Here, CRISPR/Cas9 gene editing was used to knockout HvGS3 in barley. The height of hvgs3 plants were reduced by 37.8 ~ 43.1% compared to wild type, and the culm lodging resistance index (CLRI) of the second internode of stems was increased by 76.6%. The decrease in cell length of the second internode was similar to its node length. The shorter cells may be the main reason for the declines in the internode length and plant height. The number and area of vascular bundles, the epidermal thickness, and the mechanical tissue thickness were significantly higher in hvgs3 due to the higher lignin content. Transcriptome analysis showed higher expression of structural genes related to lignin biosynthesis. Gibberellin (GA) biosynthesis was suppressed through the down-regulation of the GA3ox gene, and the application of gibberellin restored the plant height of hvgs3, indicating that plant height was altered by hindering gibberellin biosynthesis. These results shed new light on the functions of the Gγ subunit GS3 and provide a resource for breeding new lodging-resistant barley cultivars.},
}

*Hordeum/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing
*Gibberellins/metabolism
*Plant Proteins/genetics/metabolism
*Plant Stems/genetics/growth & development
*Lignin/metabolism
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Phenotype
Gene Knockout Techniques

@article {pmid39924908,
year = {2025},
author = {Gao, Y and Ang, YS and Yung, LL},
title = {CRISPR-Cas12a-Assisted DNA Circuit for Nonmicroscopic Detection of Cell Surface Receptor Clustering.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {977-985},
doi = {10.1021/acssensors.4c02770},
pmid = {39924908},
issn = {2379-3694},
mesh = {Humans ; *CRISPR-Cas Systems ; DNA/chemistry ; Cell Line, Tumor ; Receptor, ErbB-3/metabolism ; Receptor, ErbB-2/metabolism ; CRISPR-Associated Proteins/metabolism/chemistry ; Bacterial Proteins/chemistry/metabolism/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; ErbB Receptors/metabolism/genetics ; },
abstract = {Protein-protein interactions (PPIs) on the cell surface have been of great interest due to their high clinical relevance and significance; however, the methods for detecting PPIs heavily rely on microscopic instruments. In this work, we designed a Cas12a-assisted DNA circuit for detecting cell surface receptor clustering events without a dependence on microscopy. This nonmicroscopic approach is based on the proximity principle, where localized protein-protein interactions such as receptor clustering are converted into DNA barcodes. These barcodes can then be identified by Cas12a for signal generation in the bulk. The compatibility of the circuit with Cas12a was first experimentally verified. Several leak reactions were identified and minimized. Lastly, we implemented this design in human breast cancer cell line models to distinguish the different levels of human epidermal growth factor receptor 2 (HER2) homodimers and heterodimers with HER1 and HER3 semiquantitatively without the use of a microscope. Overall, our proposed Cas12a-assisted DNA circuit for detecting cell surface receptor clustering shows the potential for fast screening in diagnostic applications and drug discovery, demonstrating the promising use of enzymatic DNA circuits in biological applications.},
}

Humans
*CRISPR-Cas Systems
DNA/chemistry
Cell Line, Tumor
Receptor, ErbB-3/metabolism
Receptor, ErbB-2/metabolism
CRISPR-Associated Proteins/metabolism/chemistry
Bacterial Proteins/chemistry/metabolism/genetics
Endodeoxyribonucleases/chemistry/metabolism
ErbB Receptors/metabolism/genetics

@article {pmid39910784,
year = {2025},
author = {Pascual-Garrigos, A and Lozano-Torres, B and Das, A and Molloy, JC},
title = {Colorimetric CRISPR Biosensor: A Case Study with Salmonella Typhi.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {717-724},
doi = {10.1021/acssensors.4c02029},
pmid = {39910784},
issn = {2379-3694},
mesh = {*Colorimetry/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Salmonella typhi/genetics/isolation & purification ; beta-Galactosidase/genetics/metabolism ; Humans ; },
abstract = {There is a critical need to implement a sensitive and specific point-of-care (POC) biosensor that addresses the instrument limitations and manufacturing challenges faced in resource-constrained contexts. In this paper we focus on enteric fever which is a highly contagious and prevalent infection in low- and middle-income countries. Although easily treatable, its ambiguous symptoms paired with a lack of fast, accurate and affordable diagnostics lead to incorrect treatments which exacerbate the disease burden, including increasing antibiotic resistance. In this study, we develop a readout module for CRISPR-Cas12a that produces a colorimetric output that is visible to the naked eye and can act as a cascade signal amplifier in any CRISPR assay based on trans-cleavage. We achieve this by immobilizing an oligo covalently linked to a β-galactosidase (LacZ) enzyme, which is cleaved in the presence of DNA target-activated CRISPR-Cas12a. Upon cleavage, the colorimetric enzyme is released, and the supernatant transferred to an environment containing X-Gal producing an intense blue color. This method is capable of detecting amplified bacterial genomic DNA and has a lower limit of detection (LoD) to standard fluorescent assays while removing the requirement for costly equipment. Furthermore, it remained active 4 weeks after lyophilization, allowing for the possibility of shipment without cold chain, significantly reducing deployment costs.},
}

*Colorimetry/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Salmonella typhi/genetics/isolation & purification
beta-Galactosidase/genetics/metabolism
Humans

@article {pmid39886746,
year = {2025},
author = {Yuan, X and Geng, W and Ji, J and Yan, Z and Wei, J and Wu, Y and Yang, R},
title = {CRISPR/Cas13a-Programmed Cu NCs and Z-Scheme T-COF/Ag2S for Photoelectrochemical Biosensing of circRNA.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {1270-1279},
doi = {10.1021/acssensors.4c03180},
pmid = {39886746},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; *Copper/chemistry ; *Electrochemical Techniques/methods ; Humans ; *RNA, Circular/blood/genetics ; *Silver Compounds/chemistry ; *CRISPR-Cas Systems ; Metal-Organic Frameworks/chemistry ; Lung Neoplasms/blood ; Metal Nanoparticles/chemistry ; Limit of Detection ; Sulfides/chemistry ; Photochemical Processes ; },
abstract = {Circular RNAs (circRNAs), as a class of noncoding RNA molecules with a circular structure exhibit high stability and spatiotemporal-specific expression, making them ideal cancer biomarkers for liquid biopsy. Herein, a new photoelectrochemical (PEC) biosensor for a highly sensitive circRNA assay in the whole blood of lung cancer patients was designed based on CRISPR/Cas13a-programmed Cu nanoclusters (Cu NCs) and a Z-scheme covalent organic framework/silver sulfide (T-COF/Ag2S) composite. This Z-scheme T-COF/Ag2S composite accelerates electron transfer and produces an excellent initial photocurrent. When CRISPR/Cas13a precisely targets circRNA, it nonspecifically cleaves the triple-helix molecular structure to release DNA fragments (C'/C"). After the C'/C" opens the DNA hairpin probe (HP) modified on the electrode, hybridization chain reactions are performed to produce abundant AT-rich double-stranded DNA with the addition of H1 and H2 probes. Upon the incubation of Cu[2+], Cu NCs are in situ formed via the A-Cu[2+]-T bonds and can effectively quench the photocurrent of the Z-scheme T-COF/Ag2S due to the energy transfer process. This developed PEC biosensor for the circRNA assay shows a low limit of detection of 0.5 fM, and the reusability of DNA-modified magnetic beads (MB-DNA) reduces the detection cost. Moreover, the PEC biosensor can accurately quantify the circRNA level and distinguish the circRNA expression in whole blood from healthy controls and lung cancer patients, offering strong potential in clinical diagnosis.},
}

*Biosensing Techniques/methods
*Copper/chemistry
*Electrochemical Techniques/methods
Humans
*RNA, Circular/blood/genetics
*Silver Compounds/chemistry
*CRISPR-Cas Systems
Metal-Organic Frameworks/chemistry
Lung Neoplasms/blood
Metal Nanoparticles/chemistry
Limit of Detection
Sulfides/chemistry
Photochemical Processes

@article {pmid39862216,
year = {2025},
author = {Fu, R and Wang, Y and Qiao, S and Xu, P and Xianyu, Y and Zhang, J},
title = {CRISPR-Cas12a-Mediated Growth of Gold Nanoparticles for DNA Detection in Agarose Gel.},
journal = {ACS sensors},
volume = {10},
number = {2},
pages = {1429-1439},
doi = {10.1021/acssensors.4c03461},
pmid = {39862216},
issn = {2379-3694},
mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Sepharose/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/chemistry/genetics ; Biosensing Techniques/methods ; BRCA1 Protein/genetics ; Limit of Detection ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The rapid, simple, and sensitive detection of nucleic acid biomarkers plays a significant role in clinical diagnosis. Herein, we develop a label-free and point-of-care approach for isothermal DNA detection through the trans-cleavage activity of CRISPR-Cas12 and the growth of gold nanomaterials in agarose gel. The presence of the target can activate CRISPR-Cas12a to cleave single-stranded DNA, thus modulating the length and number of DNA sequences that mediate the growth of gold nanoparticles (AuNPs) or gold nanorods (AuNRs). Due to the extraordinary plasmonic property of gold nanomaterials, they present characteristic absorption/color after the growth with unique shapes. The sensing strategy is applied to detect BRCA-1, a biomarker related to breast cancer, with limits of detection of 1.72 pM (AuNP-based) and 2.07 pM (AuNR-based). AuNPs/AuNRs can be immobilized in agarose gels that display different colors in the presence of target DNA sequences. The agarose gel-based test allows for a readout by the naked eye or the RGB value with a smartphone. The approach is isothermal and label-free without any surface modification of nanomaterials, which holds great potential for the detection of nucleic acids in clinical applications.},
}

*Gold/chemistry
*Metal Nanoparticles/chemistry
*Sepharose/chemistry
*CRISPR-Cas Systems/genetics
Humans
DNA/chemistry/genetics
Biosensing Techniques/methods
BRCA1 Protein/genetics
Limit of Detection
CRISPR-Associated Proteins
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39715471,
year = {2025},
author = {Nguyen, NHK and Rafiee, R and Parcha, PK and Tagmount, A and Rubnitz, J and Ribeiro, R and Cao, X and Pounds, SB and Vulpe, CD and Lamba, JK},
title = {Genome-wide CRISPR/Cas9 screen identifies AraC-daunorubicin-etoposide response modulators associated with outcomes in pediatric AML.},
journal = {Blood advances},
volume = {9},
number = {5},
pages = {1078-1091},
doi = {10.1182/bloodadvances.2024014157},
pmid = {39715471},
issn = {2473-9537},
support = {T32 HG008958/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/drug therapy/mortality ; *CRISPR-Cas Systems ; *Etoposide/pharmacology/therapeutic use ; Child ; *Daunorubicin/therapeutic use/pharmacology ; *Cytarabine/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; Prognosis ; Cell Line, Tumor ; Biomarkers, Tumor ; Treatment Outcome ; },
abstract = {Cytarabine, daunorubicin, and etoposide (ADE) have been the standard backbone of induction chemotherapy regimen for patients with pediatric acute myeloid leukemia (pAML) for >5 decades. However, chemoresistance is still a major concern, and a significant proportion of pAML becomes resistant to ADE treatment and relapse, leading to poor survival. Therefore, there is a considerable need to identify mechanisms mediating drug resistance for overcoming chemoresistance. Herein, we performed synthetic lethal CRISPR/Cas9 screens using the ADE components to identify response markers. We further integrated significant markers in 3 independent pAML clinical cohorts treated with only an ADE regimen to identify drug response biomarkers with prognostic significance. We were able to identify several mediators that represent clinically and biologically significant marker genes for ADE treatment, such as BCL2, CLIP2, and VAV3, which are resistant markers to ADE, with high expression associated with poor outcomes in pAML treated with ADE, and GRPEL1, HCFC1, and TAF10, which are sensitive markers to ADE, with high expression showing beneficial outcomes. Notably, BCL2, CLIP2, and VAV3 knockdowns in their expression in AML cell lines sensitized the cells more to the ADE components, suggesting that these modulators should be further studied as potential therapeutic targets to overcome chemoresistance.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/drug therapy/mortality
*CRISPR-Cas Systems
*Etoposide/pharmacology/therapeutic use
Child
*Daunorubicin/therapeutic use/pharmacology
*Cytarabine/therapeutic use/pharmacology
Drug Resistance, Neoplasm/genetics
Antineoplastic Combined Chemotherapy Protocols/therapeutic use
Prognosis
Cell Line, Tumor
Biomarkers, Tumor
Treatment Outcome

@article {pmid39690508,
year = {2025},
author = {Wang, W and Yan, L and Li, J and Zhang, C and He, Y and Li, S and Xia, L},
title = {Engineering a robust Cas12i3 variant-mediated wheat genome editing system.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {860-873},
doi = {10.1111/pbi.14544},
pmid = {39690508},
issn = {1467-7652},
support = {2021YFF1000204//National Key Research and Development Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 2023ZD04074//STI 2030-Major Projects/ ; B23CJ0208//Hainan Seed Industry Lab/ ; //National Engineering Research Centre of Crop Molecular Breeding/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; HEK293 Cells ; Plants, Genetically Modified/genetics ; },
abstract = {Wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) is one of the most important food crops in the world. CRISPR/Cas12i3, which belongs to the type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and its less-restricted canonical 'TTN' protospacer adjacent motif (PAM). However, due to its relatively lower editing efficacy in plants and the hexaploidy complex nature of wheat, Cas12i3/Cas12i3-5M-mediated genome editing in wheat has not been documented yet. Here, we report the engineering of a robust Cas12i3-5M-mediated genome editing system in wheat through the fusion of T5 exonuclease (T5E) in combination with an optimised crRNA expression strategy (Opt). We first showed that fusion of T5E, rather than ExoI, to Cas12i3-5M increased the gene editing efficiencies by up to 1.34-fold and 3.87-fold, compared to Cas12i3-5M and Cas12i3 in HEK293T cells, respectively. However, its editing efficiency remains low in wheat. We then optimised the crRNA expression strategy and demonstrated that Opt-T5E-Cas12i3-5M could enhance the editing efficiency by 1.20- to 1.33-fold and 4.05- to 7.95-fold in wheat stable lines compared to Opt-Cas12i3-5M and Opt-Cas12i3, respectively, due to progressive 5'-end resection of the DNA strand at the cleavage site with increased deletion size. The Opt-T5E-Cas12i3-5M enabled an editing efficiency ranging from 60.71% to 90.00% across four endogenous target genes in stable lines of three elite Chinese wheat varieties. Together, the developed robust Opt-T5E-Cas12i3-5M system enriches wheat genome editing toolkits for either biological research or genetic improvement and may be extended to other important polyploidy crop species.},
}

*Triticum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Genome, Plant/genetics
CRISPR-Associated Proteins/genetics/metabolism
Humans
HEK293 Cells
Plants, Genetically Modified/genetics

@article {pmid39652457,
year = {2025},
author = {Zhao, A and Li, Q and Meng, P and Liu, P and Wu, S and Lang, Z and Song, Y and Macho, AP},
title = {Reduced content of gamma-aminobutyric acid enhances resistance to bacterial wilt disease in tomato.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {792-806},
doi = {10.1111/pbi.14539},
pmid = {39652457},
issn = {1467-7652},
support = {XDB27040204//Chinese Academy of Sciences/ ; //Chinese 1000 talents program/ ; 2022YFD2100100//National Key Research and Development Program of China/ ; },
mesh = {*Solanum lycopersicum/microbiology/genetics/metabolism ; *gamma-Aminobutyric Acid/metabolism ; *Plant Diseases/microbiology/immunology ; *Disease Resistance/genetics ; *Ralstonia solanacearum/physiology/pathogenicity ; CRISPR-Cas Systems ; Gene Editing ; Plant Roots/microbiology/genetics/metabolism ; Glutamate Decarboxylase/metabolism/genetics ; Mutation ; },
abstract = {Bacteria within the Ralstonia solanacearum species complex cause devastating diseases in numerous crops, causing important losses in food production and industrial supply. Despite extensive efforts to enhance plant tolerance to disease caused by Ralstonia, efficient and sustainable approaches are still missing. Before, we found that Ralstonia promotes the production of gamma-aminobutyric acid (GABA) in plant cells; GABA can be used as a nutrient by Ralstonia to sustain the massive bacterial replication during plant colonization. In this work, we used CRISPR-Cas9-mediated genome editing to mutate SlGAD2, which encodes the major glutamate decarboxylase responsible for GABA production in tomato, a major crop affected by Ralstonia. The resulting Slgad2 mutant plants show reduced GABA content, and enhanced tolerance to bacterial wilt disease upon Ralstonia inoculation. Slgad2 mutant plants did not show altered susceptibility to other tested biotic and abiotic stresses, including drought and heat. Interestingly, Slgad2 mutant plants showed altered microbiome composition in roots and soil. We reveal a strategy to enhance plant resistance to Ralstonia by the manipulation of plant metabolism leading to an impairment of bacterial fitness. This approach could be particularly efficient in combination with other strategies based on the manipulation of the plant immune system, paving the way to a sustainable solution to Ralstonia in agricultural systems.},
}

*Solanum lycopersicum/microbiology/genetics/metabolism
*gamma-Aminobutyric Acid/metabolism
*Plant Diseases/microbiology/immunology
*Disease Resistance/genetics
*Ralstonia solanacearum/physiology/pathogenicity
CRISPR-Cas Systems
Gene Editing
Plant Roots/microbiology/genetics/metabolism
Glutamate Decarboxylase/metabolism/genetics
Mutation

@article {pmid39630060,
year = {2025},
author = {Zhou, Y and Shi, L and Li, X and Wei, S and Ye, X and Gao, Y and Zhou, Y and Cheng, L and Cheng, L and Duan, F and Li, M and Zhang, H and Qian, Q and Zhou, W},
title = {Genetic engineering of RuBisCO by multiplex CRISPR editing small subunits in rice.},
journal = {Plant biotechnology journal},
volume = {23},
number = {3},
pages = {731-749},
doi = {10.1111/pbi.14535},
pmid = {39630060},
issn = {1467-7652},
support = {2023ZD0407203//Biological Breeding-National Science and Technology Major Project/ ; 32330079//Program of National Natural Science Foundation of China/ ; 32472040//General Program of National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/enzymology ; *Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Photosynthesis/genetics ; Plants, Genetically Modified/genetics ; Mutation/genetics ; Plant Proteins/genetics/metabolism ; Genetic Engineering/methods ; },
abstract = {Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is required for photosynthetic carbon assimilation, as it catalyses the conversion of inorganic carbon into organic carbon. Despite its importance, RuBisCO is inefficient; it has a low catalytic rate and poor substrate specificity. Improving the catalytic performance of RuBisCO is one of the key routes for enhancing plant photosynthesis. As the basic subunit of RuBisCO, RbcS affects the catalytic properties and plays a key role in stabilizing the structure of holoenzyme. Yet, the understanding of functions of RbcS in crops is still largely unknown. Toward this end, we employed CRISPR-Cas9 technology to randomly edit five rbcS genes in rice (OsrbcS1-5), generating a series of knockout mutants. The mutations of predominant rbcS genes in rice photosynthetic tissues, OsrbcS2-5, conferred inhibited growth, delayed heading and reduced yield in the field conditions, accompanying with lower RuBisCO contents and activities and significantly reduced photosynthetic efficiency. The retarded phenotypes were severer caused by multiple mutations. In addition, we revealed that these mutants had fewer chloroplasts and starch grains and a lower sugar content in the shoot base, resulting in fewer rice tillers. Further structural analysis of the mutated RuBisCO enzyme in one rbcs2,3,5 mutant line uncovered no significant differences from the wild-type protein, indicating that the mutations of rbcS did not compromise the protein assembly or the structure. Our findings generated a mutant pool with genetic diversities, which offers a valuable resource and novel insights into unravelling the mechanisms of RuBisCO in rice. The multiplex genetic engineering approach of this study provides an effective and feasible strategy for RuBisCO modification in crops, further facilitate the photosynthesis improvement and sustainable crop production.},
}

*Oryza/genetics/enzymology
*Ribulose-Bisphosphate Carboxylase/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Photosynthesis/genetics
Plants, Genetically Modified/genetics
Mutation/genetics
Plant Proteins/genetics/metabolism
Genetic Engineering/methods

@article {pmid39342963,
year = {2025},
author = {Jiang, F and Ding, X and Wang, X and Fu, K and Jia, Z and Liang, L and Guo, W},
title = {Rapid and Sensitive On-Site Nucleic Acid Detection of Three Main Fusarium Pathogens of Maize Stalk Rot Based on RPA-CRISPR/Cas12a.},
journal = {Plant disease},
volume = {109},
number = {2},
pages = {289-296},
doi = {10.1094/PDIS-08-24-1678-SR},
pmid = {39342963},
issn = {0191-2917},
mesh = {*Fusarium/genetics/isolation & purification ; *Zea mays/microbiology ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Maize stalk rot is a soilborne disease that poses a serious threat to maize production worldwide, with the most significant cause being fungal stalk rot. The development of a visual and rapid detection method for the maize stalk rot pathogen is significant for its prompt and accurate identification, enhancing agricultural production efficiency, and implementing timely preventive measures. These measures will help safeguard the maize yield and quality, ultimately reducing agricultural losses. In this study, we aimed to develop an efficient method to detect maize stalk rot pathogens. We focused on three pathogenic fungi commonly found in maize-producing regions worldwide: Fusarium verticillioides, F. proliferatum, and F. graminearum. Based on translation elongation factor 1-α, we developed a rapid detection technique using recombinase polymerase amplification-CRISPR/Cas12a, combined with test strips to develop an on-site rapid visual detection test for these pathogens. The method showed detection sensitivity for F. verticillioides, F. proliferatum, and F. graminearum within 20 min at concentrations of 7.8 pg/μl, 0.11 ng/μl, and 0.13 ng/μl, respectively. The sensitivity increased with increasing reaction time. Testing of field disease samples indicated that the method is effective in detecting nucleic acids obtained through crude extraction methods. In conclusion, we developed a visually rapid detection technology that does not rely on complex instruments and equipment for the on-site early detection of F. verticillioides, F. proliferatum, and F. graminearum in the field to implement effective control measures, ensuring stable and high maize yields.},
}

*Fusarium/genetics/isolation & purification
*Zea mays/microbiology
*Plant Diseases/microbiology
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods

@article {pmid40013512,
year = {2025},
author = {Gondalia, N and Quiroz, LF and Lai, L and Singh, AK and Khan, M and Brychkova, G and McKeown, PC and Chatterjee, M and Spillane, C},
title = {Harnessing promoter elements to enhance gene editing in plants: perspectives and advances.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.14533},
pmid = {40013512},
issn = {1467-7652},
abstract = {Genome-edited plants, endowed with climate-smart traits, have been promoted as tools for strengthening resilience against climate change. Successful plant gene editing (GE) requires precise regulation of the GE machinery, a process controlled by the promoters, which drives its transcription through interactions with transcription factors (TFs) and RNA polymerase. While constitutive promoters are extensively used in GE constructs, their limitations highlight the need for alternative approaches. This review emphasizes the promise of tissue/organ specific as well as inducible promoters, which enable targeted GE in a spatiotemporal manner with no effects on other tissues. Advances in synthetic biology have paved the way for the creation of synthetic promoters, offering refined control over gene expression and augmenting the potential of plant GE. The integration of these novel promoters with synthetic systems presents significant opportunities for precise and conditional genome editing. Moreover, the advent of bioinformatic tools and artificial intelligence is revolutionizing the characterization of regulatory elements, enhancing our understanding of their roles in plants. Thus, this review provides novel insights into the strategic use of promoters and promoter editing to enhance the precision, efficiency and specificity of plant GE, setting the stage for innovative crop improvement strategies.},
}

@article {pmid40011959,
year = {2025},
author = {Schmidt, H and Zhang, M and Chakarov, D and Bansal, V and Mourelatos, H and Sánchez-Rivera, FJ and Lowe, SW and Ventura, A and Leslie, CS and Pritykin, Y},
title = {Genome-wide CRISPR guide RNA design and specificity analysis with GuideScan2.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {41},
pmid = {40011959},
issn = {1474-760X},
support = {5T32CA160001//MSKCC TROT program/ ; P01 CA129243/NH/NIH HHS/United States ; U01 HG009395/NH/NIH HHS/United States ; U01 HG009395/NH/NIH HHS/United States ; DP2 AI171161/NH/NIH HHS/United States ; CAREER 2238831//National Science Foundation/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *CRISPR-Cas Systems ; Genome ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {We present GuideScan2 for memory-efficient, parallelizable construction of high-specificity CRISPR guide RNA (gRNA) databases and user-friendly design and analysis of individual gRNAs and gRNA libraries for targeting coding and non-coding regions in custom genomes. GuideScan2 analysis identifies widespread confounding effects of low-specificity gRNAs in published CRISPR screens and enables construction of a gRNA library that reduces off-target effects in a gene essentiality screen. GuideScan2 also enables the design and experimental validation of allele-specific gRNAs in a hybrid mouse genome. GuideScan2 will facilitate CRISPR experiments across a wide range of applications.},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics
Mice
*CRISPR-Cas Systems
Genome
Gene Editing/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40011853,
year = {2025},
author = {Sheng, T and Su, H and Yao, L and Qu, Z and Liu, H and Shao, W and Zhang, X},
title = {RhoB regulates prostate cancer cell proliferation and docetaxel sensitivity via the PI3K-AKT signaling pathway.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {354},
pmid = {40011853},
issn = {1471-2407},
mesh = {Humans ; *rhoB GTP-Binding Protein/metabolism/genetics ; *Docetaxel/pharmacology ; Male ; *Proto-Oncogene Proteins c-akt/metabolism ; *Cell Proliferation/drug effects ; *Phosphatidylinositol 3-Kinases/metabolism ; *Signal Transduction/drug effects ; *Prostatic Neoplasms/pathology/drug therapy/genetics/metabolism ; *Epithelial-Mesenchymal Transition/drug effects ; *Cell Movement/drug effects ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/therapeutic use ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {Docetaxel is a widely used first-line treatment for castration-resistant prostate cancer (CRPC). RhoB, a member of the Rho GTPase family, plays a major role in prostate cancer metastasis by modulating the PI3K-AKT signaling pathway. It is crucial in regulating cytoskeletal reassembly, cell migration, focal adhesion (FA) dynamics. To investigate RhoB's function in prostate cancer, CRISPR/Cas9 gene editing technique was utilized to knock out the RhoB gene in prostate cancer cells. Successful gene editing was confirmed by using T7 endonuclease I (T7EI) assays and Sanger sequencing. Knocking out RhoB enhanced epithelial-mesenchymal transition (EMT) and decreased the IC50 value of docetaxel in RhoB-knockout PC-3 cells. This suggests increased sensitivity to docetaxel. Furthermore, RhoB knockout prompted the migration and invasion of prostate cancer cells, effects that were reversed upon RhoB overexpression. Interestingly, RhoB status did not significantly influence the cell cycle of prostate cancer cells. RNA sequencing of PC-3 cells with either overexpressed or knock-out RhoB revealed that RhoB regulates pathways involved in FA, ECM receptor interaction, and PI3K-AKT signaling. These pathways directly influence the EMT process, cell migration, and invasion in prostate cancer cells. Notably, RhoB overexpression activated PI3K-AKT signaling when PC-3 cells were treated with low concentration of DTXL (50 nM, 72 h). This activation reduced DTXL's cytotoxicity, suggesting may confer chemoresistance via PI3K-AKT pathway activation.},
}

Humans
*rhoB GTP-Binding Protein/metabolism/genetics
*Docetaxel/pharmacology
Male
*Proto-Oncogene Proteins c-akt/metabolism
*Cell Proliferation/drug effects
*Phosphatidylinositol 3-Kinases/metabolism
*Signal Transduction/drug effects
*Prostatic Neoplasms/pathology/drug therapy/genetics/metabolism
*Epithelial-Mesenchymal Transition/drug effects
*Cell Movement/drug effects
Cell Line, Tumor
Antineoplastic Agents/pharmacology/therapeutic use
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Gene Knockout Techniques
Gene Expression Regulation, Neoplastic/drug effects

@article {pmid40011813,
year = {2025},
author = {Lukasiak, S and Kalinka, A and Gupta, N and Papadopoulos, A and Saeed, K and McDermott, U and Hannon, GJ and Ross-Thriepland, D and Walter, D},
title = {A benchmark comparison of CRISPRn guide-RNA design algorithms and generation of small single and dual-targeting libraries to boost screening efficiency.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {198},
pmid = {40011813},
issn = {1471-2164},
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Algorithms ; *Benchmarking ; Gene Library ; Genome, Human ; },
abstract = {Genome-wide CRISPR sgRNA libraries have emerged as transformative tools to systematically probe gene function. While these libraries have been iterated over time to be more efficient, their large size limits their use in some applications. Here, we benchmarked publicly available genome-wide single-targeting sgRNA libraries and evaluated dual targeting as a strategy for pooled CRISPR loss-of-function screens. We leveraged this data to design two minimal genome-wide human CRISPR-Cas9 libraries that are 50% smaller than other libraries and that preserve specificity and sensitivity, thus enabling broader deployment at scale.},
}

Humans
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Algorithms
*Benchmarking
Gene Library
Genome, Human

@article {pmid40011676,
year = {2025},
author = {Cheng, ECK and Lam, JKC and Kwon, SC},
title = {Cytosolic CRISPR RNAs for efficient application of RNA-targeting CRISPR-Cas systems.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {40011676},
issn = {1469-3178},
support = {17110223//Research Grants Council, University Grants Committee ()/ ; Start-up//University of Hong Kong (HKU)/ ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.},
}

@article {pmid40010333,
year = {2025},
author = {Keith, NC and Snyder, RA and Euler, CW and Modell, JW},
title = {Bacteria exploit viral dormancy to establish CRISPR-Cas immunity.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.02.001},
pmid = {40010333},
issn = {1934-6069},
abstract = {CRISPR-Cas systems provide prokaryotes with adaptive immunity against foreign genetic elements, including bacteriophages, by recording DNA-based immunological memories of infection called "spacers." How cells without preexisting immunity survive a rapid lytic infection long enough to acquire a new spacer and utilize it for defense remains a mystery. Here, we show that bacteria exploit the alternative dormant or "lysogenic" life cycle of temperate phages to establish CRISPR-Cas immunity. During a phage infection, immunization rates are significantly enhanced in cells entering lysogeny compared to those undergoing lysis. Furthermore, in the absence of a foreign threat, bacteria can acquire spacers targeting prophages residing within the chromosome. In this case, self-targeting by Cas9 promotes curing of the prophage, allowing immunized cells to avoid autoimmunity. The preferred acquisition of spacers during the establishment and maintenance of lysogeny may explain why most spacers in natural bacterial isolates target temperate phages.},
}

@article {pmid40008014,
year = {2025},
author = {Huang, Y and Chen, Z and Huang, H and Ding, S and Zhang, M},
title = {Important applications of DNA nanotechnology combined with CRISPR/Cas systems in biotechnology.},
journal = {RSC advances},
volume = {15},
number = {8},
pages = {6208-6230},
pmid = {40008014},
issn = {2046-2069},
abstract = {DNA nanotechnology leverages the specificity of Watson-Crick base pairing and the inherent attributes of DNA, enabling the exploitation of molecular characteristics, notably self-assembly, in nucleic acids to fabricate novel, controllable nanoscale structures and mechanisms. In the emerging field of DNA nanotechnology, DNA is not only a genetic material, but also a versatile multifunctional polymer, comprising deoxyribonucleotides, and facilitating the construction of precisely dimensioned and precise shaped two-dimensional (2D) and three-dimensional (3D) nanostructures. DNA molecules act as carriers of biological information, with notable advancements in bioimaging, biosensing, showing the profound impact. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) constitute self-defense mechanisms employed by bacteria and archaea to defend against viral invasion. With the discovery and modification of various functional Cas proteins, coupled with the identification of increasingly designable and programmable CRISPR RNAs (crRNAs), the potential of the CRISPR/Cas system in the field of molecular diagnostics is steadily being realized. Structural DNA nanotechnology provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective. This review explores biosensing detection methods that combine DNA nanotechnology with CRISPR/Cas systems. These techniques are used in biosensors to detect small molecules such as DNA, RNA, and etc. The combination of 2D and 3D DNA nanostructures with the CRISPR/Cas system holds significant value and great development prospects in the detection of important biomarkers, gene editing, and other biological applications in fields like biosensing.},
}

@article {pmid40007105,
year = {2025},
author = {Ebrecht, AC and Luelf, UJ and Govender, K and Opperman, DJ and Urlacher, VB and Smit, MS},
title = {Use of Whole Cells and Cell-Free Extracts of Catalase-Deficient E. coli for Peroxygenase-Catalyzed Reactions.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.28959},
pmid = {40007105},
issn = {1097-0290},
support = {//This work was supported by the South African Council for Scientific and Industrial Research-Industrial Biocatalysis Hub (CSIR-IBH) initiative funded by the South African Department of Science and Innovation (DSI) and the Technology Innovation Agency (TIA)./ ; },
abstract = {Unspecific peroxygenases (UPOs) and cytochrome P450 monooxygenases (CYPs) with peroxygenase activity are becoming the preferred biocatalysts for oxyfunctionalization reactions. While whole cells (WCs) or cell-free extracts (CFEs) of Escherichia coli are often preferred for cofactor-dependent monooxygenase reactions, hydrogen peroxide (H2O2) driven peroxygenase reactions are generally performed with purified enzymes, because the catalases produced by E. coli are expected to quickly degrade H2O2. We used the CRISPR/Cas system to delete the catalase encoding chromosomal genes, katG, and katE, from E. coli BL21-Gold(DE3) to obtain a catalase-deficient strain. A short UPO, DcaUPO, and two CYP peroxygenases, SscaCYP_E284A and CYP102A1_21B3, were used to compare the strains for peroxygenase expression and subsequent sulfoxidation, epoxidation, and benzylic hydroxylation activity. While 10 mM H2O2 was depleted within 10 min after addition to WCs and CFEs of the wild-type strain, at least 60% remained after 24 h in WCs and CFEs of the catalase-deficient strain. CYP peroxygenase reactions, with generally lower turnover frequencies, benefited the most from the use of the catalase-deficient strain. Comparison of purified peroxygenases in buffer versus CFEs of the catalase-deficient strain revealed that the peroxygenases in CFEs generally performed as well as the purified proteins. We also used WCs from catalase-deficient E. coli to screen three CYP peroxygenases, wild-type SscaCYP, SscaCYP_E284A, and SscaCYP_E284I for activity against 10 substrates comparing H2O2 consumption with substrate consumption and product formation. Finally, the enzyme-substrate pair with highest activity, SscaCYP_E284I, and trans-β-methylstyrene, were used in a preparative scale reaction with catalase-deficient WCs. Use of WCs or CFEs from catalase-deficient E. coli instead of purified enzymes can greatly benefit the high-throughput screening of enzyme or substrate libraries for peroxygenase activity, while they can also be used for preparative scale reactions.},
}

@article {pmid40005257,
year = {2025},
author = {Yuan, Z},
title = {From Origin to the Present: Establishment, Mechanism, Evolutions and Biomedical Applications of the CRISPR/Cas-Based Macromolecular System in Brief.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {4},
pages = {},
pmid = {40005257},
issn = {1420-3049},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; Neoplasms/genetics/therapy ; },
abstract = {Advancements in biological and medical science are intricately linked to the biological central dogma. In recent years, gene editing techniques, especially CRISPR/Cas systems, have emerged as powerful tools for modifying genetic information, supplementing the central dogma and holding significant promise for disease diagnosis and treatment. Extensive research has been conducted on the continuously evolving CRISPR/Cas systems, particularly in relation to challenging diseases, such as cancer and HIV infection. Consequently, the integration of CRISPR/Cas-based techniques with contemporary medical approaches and therapies is anticipated to greatly enhance healthcare outcomes for humans. This review begins with a brief overview of the discovery of the CRISPR/Cas system. Subsequently, using CRISPR/Cas9 as an example, a clear description of the classical molecular mechanism underlying the CRISPR/Cas system was given. Additionally, the development of the CRISPR/Cas system and its applications in gene therapy and high-sensitivity disease diagnosis were discussed. Furthermore, we address the prospects for clinical applications of CRISPR/Cas-based gene therapy, highlighting the ethical considerations associated with altering genetic information. This brief review aims to enhance understanding of the CRISPR/Cas macromolecular system and provide insight into the potential of genetic macromolecular drugs for therapeutic purposes.},
}

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

@article {pmid40004557,
year = {2025},
author = {Wang, J and Wang, H and Zhai, J and Zhu, F and Ren, Y and Zhou, J and Zhang, Z and Luo, L and Xu, W},
title = {Identification of Ziziphus jujuba cv. Dongzao DNA Demethylase ZjROS1 Gene Family and Construction of CRISPR/Cas9-Mediated Gene-Editing Vector.},
journal = {Genes},
volume = {16},
number = {2},
pages = {},
pmid = {40004557},
issn = {2073-4425},
support = {YCX24138//Graduate Innovation Program of North Minzu University/ ; 2022QCXTD04//Innovation Team for Genetic Improvement of Economic Forests/ ; 32060669//National Natural Science Foundation of China/ ; },
mesh = {*Ziziphus/genetics/growth & development ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; DNA Methylation/genetics ; Fruit/genetics/growth & development ; Multigene Family ; },
abstract = {DNA methylation is one of the earliest and most extensively studied epigenetic regulatory mechanisms. The ROS1 (Repressor of Silencing 1) gene was first discovered in Arabidopsis thaliana, and it is a DNA demethylase that can remove 5-methylcytosine from DNA, thereby affecting DNA methylation levels and gene expression. Objectives: The objective of this study was to investigate the role of ROS1 in the development and maturation of Ziziphus jujuba cv. "Dongzao" fruit. Methods: We cloned the ROS1 gene and conducted bioinformatics and expression characteristics analyses on it. Results: Three ROS1 genes, named ZjROS1-1~3, was identified, and each member protein was localized in the nucleus, cytoskeleton, chloroplast, and vacuole. The promoter contained cis-elements such as light response, plant hormone signal transduction, and stress response cis-elements, and it interacted with many proteins such as CMT, MET, and ZDP. The results of the real-time fluorescence quantitative PCR show that ZjROS1 has specific expression patterns in different tissues of Z. jujuba cv. Dongzao, and the expression of ZjROS1-2 in flowers and fruits is high. At the same time, CRISPR/Cas9 technology was used to construct a gene-editing vector for ZjROS1, which provided a basis for the subsequent genetic transformation. Conclusions: In this study, the biological function of ZjROS1 was clarified and a gene-editing vector was constructed, which provided a theoretical basis for the regulation mechanism of demethylase ZjROS1 in the fruit ripening and development of Z. jujuba cv. Dongzao.},
}

*Ziziphus/genetics/growth & development
*Gene Editing/methods
*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
DNA Methylation/genetics
Fruit/genetics/growth & development
Multigene Family

@article {pmid39954255,
year = {2025},
author = {Xiang, RR and Lee, SA and Tyndall, CF and Bhatia, AR and Yin, J and Singler, C and Hauk, BJ and Kipp, MP and Takeda, DY},
title = {CRISPR screening identifies regulators of enhancer-mediated androgen receptor transcription in advanced prostate cancer.},
journal = {Cell reports},
volume = {44},
number = {2},
pages = {115312},
doi = {10.1016/j.celrep.2025.115312},
pmid = {39954255},
issn = {2211-1247},
mesh = {Male ; *Receptors, Androgen/metabolism/genetics ; Humans ; *Homeodomain Proteins/metabolism/genetics ; *Enhancer Elements, Genetic/genetics ; *Hepatocyte Nuclear Factor 3-alpha/metabolism/genetics ; *GATA2 Transcription Factor/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; Cell Line, Tumor ; Prostatic Neoplasms, Castration-Resistant/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Transcription Factor AP-2/metabolism/genetics ; Prostatic Neoplasms/genetics/pathology/metabolism ; Transcription, Genetic ; Mice ; Protein Binding ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Amplification of the androgen receptor (AR) locus is the most frequent alteration in metastatic castration-resistant prostate cancer (CRPC). Recently, it was discovered that an enhancer of the AR is co-amplified with the AR gene body and contributes to increased AR transcription and resistance to androgen deprivation therapy. However, the mechanism of enhancer activation in advanced disease is unknown. Here, we used CRISPR-Cas9 screening to identify transcription factors that bind to the AR enhancer and modulate enhancer-mediated AR transcription. We demonstrate that HOXB13, GATA2, and TFAP2C bind the AR enhancer in patient-derived xenografts and directly impact features associated with an active chromatin state. Interestingly, the AR enhancer belongs to a set of regulatory elements that require HOXB13 to maintain FOXA1 binding, further delineating the role of HOXB13 in CRPC. This work provides a framework to functionally identify trans-acting factors required for the activation of disease-related noncoding regulatory elements.},
}

Male
*Receptors, Androgen/metabolism/genetics
Humans
*Homeodomain Proteins/metabolism/genetics
*Enhancer Elements, Genetic/genetics
*Hepatocyte Nuclear Factor 3-alpha/metabolism/genetics
*GATA2 Transcription Factor/metabolism/genetics
*CRISPR-Cas Systems/genetics
Animals
Cell Line, Tumor
Prostatic Neoplasms, Castration-Resistant/genetics/pathology/metabolism
Gene Expression Regulation, Neoplastic
Transcription Factor AP-2/metabolism/genetics
Prostatic Neoplasms/genetics/pathology/metabolism
Transcription, Genetic
Mice
Protein Binding
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39936934,
year = {2025},
author = {Lu, C and Chen, J and Zhang, S and Tian, Y and Ying, X and Zhang, D and Luo, Z and Si, X and Li, M},
title = {Development of a vitrified CRISPR/Cas12b-based assay for rapid genotyping of SLCO1B1 SNPs without DNA amplification.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {9},
pages = {2083-2093},
doi = {10.1039/d4ay02015d},
pmid = {39936934},
issn = {1759-9679},
mesh = {*Polymorphism, Single Nucleotide/genetics ; Humans ; *Liver-Specific Organic Anion Transporter 1/genetics ; *CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Two single nucleotide polymorphisms (SNPs) in the human SLCO1B1 gene, c.388A>G (rs2306283) and c.521T>C (rs4149056), are independent determinants of the efficacy and side effects of statin drugs. Multinational clinical guidelines recommend testing for SLCO1B1 genotypes before the initial use of statins. Current SLCO1B1 SNP identification methods, primarily based on quantitative fluorescence PCR, rely on expensive equipment, are time-consuming, and require cold-chain storage for reagents, making them unsuitable for use in resource-limited healthcare settings. In this study, we developed a CRISPR/Cas12b-based amplification-free genotyping technique for SLCO1B1 SNPs. Within 30 minutes of the isothermal reaction, genotyping of the c.388A>G and c.521T>C SNPs in the SLCO1B1 gene can be observed by the naked eyes under blue light. Additionally, maltodextrin was identified as an effective vitrification stabilizer for the CRISPR/Cas12b premix. A low-cost vitrification process was optimized to prepare a glass like solid reagent via room-temperature vacuum drying. The vitrified CRISPR/Cas12b reagent retained approximately 88% of its activity after 30 days of storage at 37 °C, eliminating the need for cold-chain storage and allowing for long-term preservation at room temperature. This vitrified CRISPR/Cas12b based rapid SNP detection technique is especially suitable for genotyping drug metabolism genes in primary healthcare settings, providing effective guidance for precision medicine in clinical practice.},
}

*Polymorphism, Single Nucleotide/genetics
Humans
*Liver-Specific Organic Anion Transporter 1/genetics
*CRISPR-Cas Systems/genetics
*Genotyping Techniques/methods
Genotype
Nucleic Acid Amplification Techniques/methods

@article {pmid39930995,
year = {2025},
author = {Nafian, F and Esfahani, KS and Hobabi Aghmiuni, M and Khoushab, S and Illeslamllo, T and Nafian, S and Mohamadiyan, N and Aleyasin, NS and Kamali Doust Azad, B},
title = {Emerging microfluidic technologies for CRISPR-based diagnostics: an overview.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {9},
pages = {1962-1976},
doi = {10.1039/d5ay00063g},
pmid = {39930995},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microfluidic Analytical Techniques/methods/instrumentation ; Point-of-Care Testing ; Lab-On-A-Chip Devices ; },
abstract = {In recent years, CRISPR (clustered regularly interspaced short palindromic repeats) has emerged as a detection technique with high specificity and sensitivity. However, it still needs improvements in terms of reducing cost, complexity, cross-contamination, technical requirements, and lack of quantification platforms. Microfluidic strategies can advance CRISPR-based technology and be modified to a higher level in the future. This review provides an overview of CRISPR-based detection systems (CRISPR-Dx) and their mechanism. Then, it explains how they have been optimized for fast and accurate point-of-care testing (POCT) using microfluidic devices such as SHINE, CARMEN, DNAiTECH, Dμchip, MAPnavi, FAST, and ITP. We discuss their innovations, primarily focusing on how they develop CRISPR-Dx in detection throughput, quantification, simple operation, visualization, sensitivity, specificity, and anti-contamination.},
}

Humans
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Microfluidic Analytical Techniques/methods/instrumentation
Point-of-Care Testing
Lab-On-A-Chip Devices

@article {pmid39838098,
year = {2025},
author = {Whalen, JM and Earley, J and Wisniewski, C and Mercurio, AM and Cantor, SB},
title = {Targeting BRCA1-deficient PARP inhibitor-resistant cells with nickases reveals nick resection as a cancer vulnerability.},
journal = {Nature cancer},
volume = {6},
number = {2},
pages = {278-291},
pmid = {39838098},
issn = {2662-1347},
support = {R01 CA254037/CA/NCI NIH HHS/United States ; 1F32CA268524-01A1//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; R01CA285607//U.S. Department of Health & Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; R01 CA254037/CA/NCI NIH HHS/United States ; R01 CA254037/CA/NCI NIH HHS/United States ; },
mesh = {*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; *BRCA1 Protein/genetics/deficiency ; Animals ; *Tumor Suppressor p53-Binding Protein 1/genetics/metabolism ; Humans ; Mice ; *Drug Resistance, Neoplasm/genetics ; Female ; Cell Line, Tumor ; Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/genetics/metabolism ; Homologous Recombination ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics ; Breast Neoplasms/genetics/drug therapy ; },
abstract = {Tumors lacking the BRCA1 and BRCA2 (BRCA) hereditary breast cancer genes display heightened sensitivity to anti-cancer treatments, such as inhibitors of poly (ADP-ribose) polymerase 1 (PARP1). However, when resistance develops, treatments are lacking. Using CRISPR technology, we discovered that enhancing homologous recombination through increased DNA end resection in BRCA1-deficient cells by loss of the 53BP1-Shieldin complex-which is associated with resistance to PARP inhibitors-also heightens sensitivity to DNA nicks. The sensitivity is caused by hyper-resection of nicks into extensive single-stranded regions that trigger cell death. Based on these findings and that nicks limit tumor formation in mice, we propose nickases as a tool for personalized medicine. Moreover, our findings indicate that restricting nick expansion is a critical function of the 53BP1-Shieldin complex.},
}

*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
*BRCA1 Protein/genetics/deficiency
Animals
*Tumor Suppressor p53-Binding Protein 1/genetics/metabolism
Humans
Mice
*Drug Resistance, Neoplasm/genetics
Female
Cell Line, Tumor
Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors/genetics/metabolism
Homologous Recombination
CRISPR-Cas Systems
DNA-Binding Proteins/genetics
Breast Neoplasms/genetics/drug therapy

@article {pmid39716488,
year = {2025},
author = {He, Y and Zalenski, N and Stephenson, AA and Raper, AT and Ghimire, C and Suo, Z},
title = {Conformational transitions of Streptococcus pyogenes Cas9 induced by salt and single-guide RNA binding.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {2},
pages = {108120},
pmid = {39716488},
issn = {1083-351X},
support = {R01 GM122093/GM/NIGMS NIH HHS/United States ; },
mesh = {*Streptococcus pyogenes/enzymology/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *CRISPR-Associated Protein 9/metabolism/chemistry ; *Potassium Chloride/pharmacology/metabolism/chemistry ; Protein Conformation ; Bacterial Proteins/metabolism/chemistry/genetics ; Fluorescence Resonance Energy Transfer ; },
abstract = {Streptococcus pyogenes (Sp) Cas9 has been widely utilized to edit genomes across diverse species. To achieve high efficiency and specificity as a gene-editing enzyme, Sp Cas9 undergoes a series of sequential conformational changes during substrate binding and catalysis. Here, we employed single-molecule FRET techniques to investigate the effect of different KCl concentrations on conformational dynamics of Sp Cas9 in the presence or the absence of a single-guide RNA (sgRNA). In the absence of sgRNA and at low KCl concentrations (75 mM), apo Cas9 surprisingly exhibited two distinct conformations: a primary autoinhibited open conformation (apo Cas9 conformation [Cas9[apo]]) and a secondary sgRNA-bound-like conformation (Cas9[X]). Interestingly, increase in buffer KCl concentration led to a linear increase in the Cas9[X] population and a corresponding decrease in the Cas9[apo] population. In contrast, changes in KCl concentration exerted the opposite effects on the Cas9[X] and Cas9[apo] populations in the presence of sgRNA. Collectively, our findings by using KCl concentration as the probe suggest that Cas9 might employ a conformational sampling mechanism, in addition to the more common induced-fit mechanism established by us previously, for sgRNA binding.},
}

*Streptococcus pyogenes/enzymology/metabolism
*RNA, Guide, CRISPR-Cas Systems/metabolism
*CRISPR-Associated Protein 9/metabolism/chemistry
*Potassium Chloride/pharmacology/metabolism/chemistry
Protein Conformation
Bacterial Proteins/metabolism/chemistry/genetics
Fluorescence Resonance Energy Transfer

@article {pmid39266721,
year = {2025},
author = {Caliendo, F and Vitu, E and Wang, J and Kuo, SH and Sandt, H and Enghuus, CN and Tordoff, J and Estrada, N and Collins, JJ and Weiss, R},
title = {Customizable gene sensing and response without altering endogenous coding sequences.},
journal = {Nature chemical biology},
volume = {21},
number = {3},
pages = {348-359},
pmid = {39266721},
issn = {1552-4469},
support = {W911NF19C0008//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; HR0011-20-2-0005//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; 5-R01-EB030946-04//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; 5-R01-EB025256-04//U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; 5R01HD105947-03//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; CNS-1446474//National Science Foundation (NSF)/ ; W911NF-17-3-003//United States Department of Defense | United States Army | U.S. Army Research, Development and Engineering Command | Army Research Office (ARO)/ ; 5RC2DK120535//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; },
mesh = {Animals ; CHO Cells ; *Cricetulus ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Synthetic Biology/methods ; Cell Survival ; },
abstract = {Synthetic biology aims to modify cellular behaviors by implementing genetic circuits that respond to changes in cell state. Integrating genetic biosensors into endogenous gene coding sequences using clustered regularly interspaced short palindromic repeats and Cas9 enables interrogation of gene expression dynamics in the appropriate chromosomal context. However, embedding a biosensor into a gene coding sequence may unpredictably alter endogenous gene regulation. To address this challenge, we developed an approach to integrate genetic biosensors into endogenous genes without modifying their coding sequence by inserting into their terminator region single-guide RNAs that activate downstream circuits. Sensor dosage responses can be fine-tuned and predicted through a mathematical model. We engineered a cell stress sensor and actuator in CHO-K1 cells that conditionally activates antiapoptotic protein BCL-2 through a downstream circuit, thereby increasing cell survival under stress conditions. Our gene sensor and actuator platform has potential use for a wide range of applications that include biomanufacturing, cell fate control and cell-based therapeutics.},
}

Animals
CHO Cells
*Cricetulus
*Biosensing Techniques/methods
CRISPR-Cas Systems
Proto-Oncogene Proteins c-bcl-2/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Expression Regulation
Synthetic Biology/methods
Cell Survival

@article {pmid38828264,
year = {2024},
author = {Ayllon-Hermida, A and Nicolau-Fernandez, M and Larrinaga, AM and Aparici-Herraiz, I and Tintó-Font, E and Llorà-Batlle, O and Orban, A and Yasnot, MF and Graupera, M and Esteller, M and Popovici, J and Cortés, A and Del Portillo, HA and Fernandez-Becerra, C},
title = {Plasmodium vivax spleen-dependent protein 1 and its role in extracellular vesicles-mediated intrasplenic infections.},
journal = {Frontiers in cellular and infection microbiology},
volume = {14},
number = {},
pages = {1408451},
pmid = {38828264},
issn = {2235-2988},
mesh = {*Plasmodium vivax/genetics/immunology/metabolism/pathogenicity ; *Protozoan Proteins/genetics/immunology/metabolism ; *Spleen/cytology/immunology/parasitology/pathology ; *Extracellular Vesicles/immunology/metabolism/parasitology ; Reticulocytes/cytology/parasitology ; Fibroblasts/parasitology ; Gene Expression Regulation ; CRISPR-Cas Systems ; Genome, Protozoan ; Single-Cell Gene Expression Analysis ; *Malaria, Vivax/blood/immunology/parasitology/pathology ; Humans ; Cell Adhesion/immunology ; Colombia ; Antibodies, Protozoan/immunology/metabolism ; Primary Cell Culture ; Host-Parasite Interactions/immunology ; Male ; Female ; Mice ; },
abstract = {Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax. Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax-infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax, a major challenge for malaria elimination.},
}

*Plasmodium vivax/genetics/immunology/metabolism/pathogenicity
*Protozoan Proteins/genetics/immunology/metabolism
*Spleen/cytology/immunology/parasitology/pathology
*Extracellular Vesicles/immunology/metabolism/parasitology
Reticulocytes/cytology/parasitology
Fibroblasts/parasitology
Gene Expression Regulation
CRISPR-Cas Systems
Genome, Protozoan
Single-Cell Gene Expression Analysis
*Malaria, Vivax/blood/immunology/parasitology/pathology
Humans
Cell Adhesion/immunology
Colombia
Antibodies, Protozoan/immunology/metabolism
Primary Cell Culture
Host-Parasite Interactions/immunology
Male
Female
Mice

@article {pmid40004545,
year = {2025},
author = {Shu, H and Luan, A and Ullah, H and He, J and Wang, Y and Chen, C and Wei, Q and Zhan, R and Chang, S},
title = {Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.},
journal = {Genes},
volume = {16},
number = {2},
pages = {},
doi = {10.3390/genes16020217},
pmid = {40004545},
issn = {2073-4425},
support = {32272677//National Natural Science Foundation, China/ ; 1630032024001//Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences/ ; },
mesh = {*Ananas/genetics/enzymology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {BACKGROUND/OBJECTIVES: CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)-associated protein 9 is now widely used in agriculture and medicine. Off-target effects can lead to unexpected results that may be harmful, and these effects are a common concern in both research and therapeutic applications.

METHODS: In this study, using pineapple as the gene-editing material, eighteen target sequences with varying numbers of PAM (Protospacer-Adjacent Motif) sites were used to construct gRNA vectors. Fifty mutant lines were generated for each target sequence, and the off-target rates were counted.

RESULTS: Selecting sequences with multiple flanking PAM sites as editing targets resulted in a lower off-target rate compared to those with a single PAM site. Target sequences with two 5'-NGG ("N" represents any nucleobase, followed by two guanine "G") PAM sites at the 3' end exhibited greater specificity and a higher probability of binding with the Cas9 protein than those only with one 5'-NGG PAM site at the 3' end. Conversely, although the target sequence with a 5'-NAG PAM site (where "N" is any nucleobase, followed by adenine "A" and guanine "G") adjacent and upstream of an NGG PAM site had a lower off-target rate compared to sequences with only an NGG PAM site, their off-target rates were still higher than those of sequences with two adjacent 5'-NAG PAM sites. Among the target sequences of pineapple mutant lines (AcACS1, AcOT5, AcCSPE6, AcPKG11A), more deletions than insertions were found.

CONCLUSIONS: We found that target sequences with multiple flanking PAM sites are more likely to bind with the Cas9 protein and induce mutations. Selecting sequences with multiple flanking PAM sites as editing targets can reduce the off-target effects of the Cas9 enzyme in pineapple. These findings provide a foundation for improving off-target prediction and engineering CRISPR-Cas9 complexes for gene editing.},
}

*Ananas/genetics/enzymology
*CRISPR-Cas Systems
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid40004462,
year = {2025},
author = {Lara, P and Aguilar-González, A and Martín, F and Mesas, C and Moreno, J and Rama, AR},
title = {Exploring miR-21 Knock-Out Using CRISPR/Cas as a Treatment for Lung Cancer.},
journal = {Genes},
volume = {16},
number = {2},
pages = {},
doi = {10.3390/genes16020133},
pmid = {40004462},
issn = {2073-4425},
mesh = {Humans ; *MicroRNAs/genetics ; *Lung Neoplasms/genetics/drug therapy/pathology ; *CRISPR-Cas Systems ; *PTEN Phosphohydrolase/genetics ; A549 Cells ; *Cell Proliferation/genetics ; *Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic ; Apoptosis Regulatory Proteins/genetics ; Cell Movement/genetics ; Gene Knockout Techniques/methods ; RNA-Binding Proteins/genetics ; Antineoplastic Agents/pharmacology ; Paclitaxel/pharmacology ; Carboplatin/pharmacology ; Gemcitabine ; Gene Editing/methods ; Deoxycytidine/analogs & derivatives/pharmacology ; },
abstract = {BACKGROUND: Lung cancer is a leading cause of cancer-related deaths worldwide. Its high incidence and poor prognosis demonstrate the need to investigate new therapies. The PI3K/AKT pathway is activated in carcinogenic processes such as invasion, proliferation, and drug resistance. MiR-21 is a microRNA overexpressed in numerous types of cancer and which activates PI3K/AKT pathway by down-regulating its main targets, PTEN and PDCD4. CRISPR is a revolutionary gene-editing technology that allows genes to be deleted. The aim of this study was to use CRISPR/Cas9 technology as an option to reduce carcinogenic and drug resistance processes by eliminating miR-21.

METHODS: CRISPR/Cas9 was used to knock out miR-21 (miR-21 KO) in A549 lung cancer cells and thus reverse the carcinogenic processes activated by miR-21 overexpression. Furthermore, the effect of miR-21 KO on drug resistance was studied, choosing the main chemotherapeutic agents used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.

RESULTS: miR-21 KO A549 cells exhibited a reduction in proliferation, migration, and colony formation compared to A549 cells. In contrast, the expression of PTEN and PDCD4 increased in miR-21 KO A549 cells. Furthermore, miR-21 KO A549 cells showed a decrease in the IC50 of the drugs used for the treatment of lung cancer: gemcitabine, carboplatin, paclitaxel, and oxaliplatin.

CONCLUSIONS: Based on these results, miR-21 knock-out using CRISPR/Cas could be a promising strategy for the treatment of lung cancer.},
}

Humans
*MicroRNAs/genetics
*Lung Neoplasms/genetics/drug therapy/pathology
*CRISPR-Cas Systems
*PTEN Phosphohydrolase/genetics
A549 Cells
*Cell Proliferation/genetics
*Drug Resistance, Neoplasm/genetics
Gene Expression Regulation, Neoplastic
Apoptosis Regulatory Proteins/genetics
Cell Movement/genetics
Gene Knockout Techniques/methods
RNA-Binding Proteins/genetics
Antineoplastic Agents/pharmacology
Paclitaxel/pharmacology
Carboplatin/pharmacology
Gemcitabine
Gene Editing/methods
Deoxycytidine/analogs & derivatives/pharmacology

@article {pmid40004186,
year = {2025},
author = {Nakamae, K and Suzuki, T and Yonezawa, S and Yamamoto, K and Kakuzaki, T and Ono, H and Naito, Y and Bono, H},
title = {Risk Prediction of RNA Off-Targets of CRISPR Base Editors in Tissue-Specific Transcriptomes Using Language Models.},
journal = {International journal of molecular sciences},
volume = {26},
number = {4},
pages = {},
doi = {10.3390/ijms26041723},
pmid = {40004186},
issn = {1422-0067},
support = {030RP2022//ROIS-DS-JOINT 2022/ ; 009RP2023//ROIS-DS-JOINT 2023/ ; 012RP2024//ROIS-DS-JOINT 2024/ ; JPMJPF2010//The Center of Innovation for Bio-Digital Transformation (BioDX), an open innovation platform for industry-academia cocreation (COI-NEXT) and the Japan Science and Technology Agency (JST)/ ; },
mesh = {*Transcriptome/genetics ; Humans ; *Machine Learning ; *Gene Editing/methods ; CRISPR-Cas Systems ; RNA Editing/genetics ; RNA/genetics ; Organ Specificity/genetics ; Computational Biology/methods ; },
abstract = {Base-editing technologies, particularly cytosine base editors (CBEs), allow precise gene modification without introducing double-strand breaks; however, unintended RNA off-target effects remain a critical concern and are under studied. To address this gap, we developed the Pipeline for CRISPR-induced Transcriptome-wide Unintended RNA Editing (PiCTURE), a standardized computational pipeline for detecting and quantifying transcriptome-wide CBE-induced RNA off-target events. PiCTURE identifies both canonical ACW (W = A or T/U) motif-dependent and non-canonical RNA off-targets, revealing a broader WCW motif that underlies many unanticipated edits. Additionally, we developed two machine learning models based on the DNABERT-2 language model, termed STL and SNL, which outperformed motif-only approaches in terms of accuracy, precision, recall, and F1 score. To demonstrate the practical application of our predictive model for CBE-induced RNA off-target risk, we integrated PiCTURE outputs with the Predicting RNA Off-target compared with Tissue-specific Expression for Caring for Tissue and Organ (PROTECTiO) pipeline and estimated RNA off-target risk for each transcript showing tissue-specific expression. The analysis revealed differences among tissues: while the brain and ovaries exhibited relatively low off-target burden, the colon and lungs displayed relatively high risks. Our study provides a comprehensive framework for RNA off-target profiling, emphasizing the importance of advanced machine learning-based classifiers in CBE safety evaluations and offering valuable insights to inform the development of safer genome-editing therapies.},
}

*Transcriptome/genetics
Humans
*Machine Learning
*Gene Editing/methods
CRISPR-Cas Systems
RNA Editing/genetics
RNA/genetics
Organ Specificity/genetics
Computational Biology/methods

@article {pmid40004028,
year = {2025},
author = {Zeng, M and Zhang, QY and Ke, F},
title = {Characterization of Five CRISPR Systems in Microcystis aeruginosa FACHB-524 with Focus on the In Vitro Antiviral Activity of One CRISPR System.},
journal = {International journal of molecular sciences},
volume = {26},
number = {4},
pages = {},
doi = {10.3390/ijms26041554},
pmid = {40004028},
issn = {1422-0067},
support = {2018YFA0903101//the National Key R&D Programs of China/ ; },
mesh = {*Microcystis/genetics/virology ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteriophages/genetics ; },
abstract = {Microcystis aeruginosa is an important species causing cyanobacterial blooms, which can be effectively infected and lysed by cyanophages. Several strategies have been developed by M. aeruginosa to resist cyanophage infections, including the CRISPR-Cas systems. However, detailed information on the CRISPR-Cas systems in M. aeruginosa is rare. In the present study, the CRISPR-Cas systems of M. aeruginosa FACHB-524 were analyzed by genome re-sequencing, which showed that there are two type I (Cluster 1, I-B1; Cluster 2, I-D) and three type III-B (Cluster 3/4/5) CRISPR-Cas systems in the cyanobacteria. Further comparison revealed that spacer sequences of two type III-B systems targeted several genes of the cyanophage MaMV (M. aeruginosa myovirus) strains. One of the type III systems (Cluster 4) was then cloned and expressed in Escherichia coli BL21 (DE3). Protein purification and mass spectrometry identification revealed that a Cmr-crRNA effector complex formed in the E. coli. Subsequently, T4 phage (T4) was used to infect the E. coli, expressing the Cmr-crRNA complex with or without accessory proteins. The results showed that the Cmr-crRNA effector complex exhibited anti-phage activity and the accessory protein Csx1 enhanced the immune activity of the complex. Collectively, our results comprehensively demonstrate the CRISPR systems encoded by a strain of M. aeruginosa, and for the first time, one of the CRISPR systems was constructed into E. coli, providing a foundation for further in-depth analysis of cyanobacterial CRISPR systems.},
}

*Microcystis/genetics/virology
*CRISPR-Cas Systems
Escherichia coli/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacteriophages/genetics

@article {pmid40003981,
year = {2025},
author = {Asad, M and Chang, Y and Liao, J and Yang, G},
title = {CRISPR/Cas9 Genome Editing in the Diamondback Moth: Current Progress, Challenges, and Prospects.},
journal = {International journal of molecular sciences},
volume = {26},
number = {4},
pages = {},
doi = {10.3390/ijms26041515},
pmid = {40003981},
issn = {1422-0067},
support = {2023J02009//Key Project of Fujian Province Natural Science Foundation/ ; 32072425//Natural Science Foundation of China/ ; 2018NZ01010013//Special Key Project of Fujian Province, grant number/ ; KRA16001A//111" program/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Moths/genetics ; Insecticide Resistance/genetics ; },
abstract = {The development of site-specific genome-editing tools like CRISPR (clustered regularly interspaced short palindromic repeat) and its associated protein, Cas9, is revolutionizing genetic engineering with its highly efficient mechanism, offering the potential for effective pest management. Recently, CRISPR/Cas9 gene-editing has been extensively utilized in the management of the diamondback moth, Plutella xylostella (L.), a highly destructive pest of vegetable crops, for different purposes, such as gene function analysis and genetic control. However, the progress related to this gene-editing tool in P. xylostella has not yet been summarized. This review highlights the progress and applications of CRISPR/Cas9 in uncovering the genes critical for development, reproduction, and insecticide resistance in P. xylostella. Moreover, the progress related to the CRISPR/Cas9 gene drive for population suppression and modifications has also been discussed. In addition to the significant progress made, challenges such as low germline editing efficiency and limited homology-directed repair remain obstacles to its widespread application. To address these limitations, we have discussed the different strategies that are anticipated to improve the efficiency of CRISPR/Cas9, paving the way to it becoming a pivotal tool in sustainable pest management. Therefore, the present review will help researchers in the future enhance the efficiency of the CRISPR/Cas9 system and use it to manage the diamondback moth.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Moths/genetics
Insecticide Resistance/genetics

@article {pmid40003953,
year = {2025},
author = {Zhao, M and Yang, Z and Li, J and Ming, F and Kong, D and Xu, H and Wang, Y and Chen, P and Duan, X and Wang, M and Wang, Z},
title = {Enhancement of Dendrobine Production by CRISPR/Act3.0-Mediated Transcriptional Activation of Multiple Endogenous Genes in Dendrobium Plants.},
journal = {International journal of molecular sciences},
volume = {26},
number = {4},
pages = {},
doi = {10.3390/ijms26041487},
pmid = {40003953},
issn = {1422-0067},
support = {JCYJ20210324123005016, JCYJ20230807151000001//The Science, Technology and Innovation Commission of Shenzhen Municipality/ ; },
mesh = {*Dendrobium/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Plants, Genetically Modified/genetics ; *Transcriptional Activation ; Diterpenes/metabolism ; Alkaloids ; },
abstract = {Dendrobine, a significant medicinal compound, typically accumulates at low concentrations within several Dendrobium species, including Dendrobium nobile, Dendrobium catenatum, and Dendrobium moniliforme. As D. nobile and D. catenatum are established ingredients in traditional Chinese medicine and have been cultivated extensively, they present ideal plant chassis for upscaling dendrobine production for industrial and research applications. This study employed two approaches: the ectopic overexpression of seven genes through multigene stacking and the activation of multiple key endogenous genes in the dendrobine synthesis pathway using CRISPR/Act3.0 in either D. nobile or D. catenatum. These methods enhanced dendrobine production in transiently infiltrated leaves by 30.1% and transgenic plants by 35.6%. The study is the first to apply CRISPR/Act3.0 to Dendrobium orchids, enhancing dendrobine production, and thus laying a solid foundation for further improvements. CRISPR/Act3.0 is a recently developed technique that demonstrates high efficiency in model plant species, including rice, maize, and Arabidopsis. By combining CRISPR with transcriptional regulatory modules, activation of multiple endogenous genes in the metabolic pathway can be achieved. The successful application in orchid molecular breeding reveals promising potential for further exploration.},
}

*Dendrobium/genetics/metabolism
*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
*Plants, Genetically Modified/genetics
*Transcriptional Activation
Diterpenes/metabolism
Alkaloids

@article {pmid40003875,
year = {2025},
author = {Ilchuk, LA and Kochegarova, KK and Baikova, IP and Safonova, PD and Bruter, AV and Kubekina, MV and Okulova, YD and Minkovskaya, TE and Kuznetsova, NA and Dolmatova, DM and Ryabinina, AY and Mozhaev, AA and Belousov, VV and Ershov, BP and Timashev, PS and Filatov, MA and Silaeva, YY},
title = {Mutations in Filamin C Associated with Both Alleles Do Not Affect the Functioning of Mice Cardiac Muscles.},
journal = {International journal of molecular sciences},
volume = {26},
number = {4},
pages = {},
doi = {10.3390/ijms26041409},
pmid = {40003875},
issn = {1422-0067},
support = {075-15-2019-1661//The Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Animals ; *Filamins/genetics/metabolism ; Mice ; *Alleles ; *Mutation ; Myocardium/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Heterozygote ; },
abstract = {Filamin C (FLNC) is a structural protein of muscle fibers. Mutations in the FLNC gene are known to cause myopathies and cardiomyopathies in humans. Here we report the generation by a CRISPR/Cas9 editing system injected into zygote pronuclei of two mouse strains carrying filamin C mutations-one of them (AGA) has a deletion of three nucleotides at position c.7418_7420, causing E>>D substitution and N deletion at positions 2472 and 2473, respectively. The other strain carries a deletion of GA nucleotides at position c.7419_7420, leading to a frameshift and a premature stop codon. Homozygous animals (Flnc[AGA/AGA] and Flnc[GA/GA]) were embryonically lethal. We determined that Flnc[GA/GA] embryos died prior to the E12.5 stage and illustrated delayed development after the E9.5 stage. We performed histological analysis of heart tissue and skeletal muscles of heterozygous strains carrying mutations in different combinations (Flnc[GA/wt], Flnc[AGA/wt], and Flnc[GA/AGA]). By performing physiological tests (grip strength and endurance tests), we have shown that heterozygous animals of both strains (Flnc[GA/wt], Flnc[AGA/wt]) are functionally indistinguishable from wild-type animals. Interestingly, compound heterozygous mice (Flnc[GA/AGA]) are viable, develop normally, reach puberty and it was verified by ECG and Eco-CG that their cardiac muscle is functionally normal. Intriguingly, Flnc[GA/AGA] mice demonstrated better results in the grip strength physiological test in comparison to WT animals. We also propose a structural model that explains the complementary interaction of two mutant variants of filamin C.},
}

Animals
*Filamins/genetics/metabolism
Mice
*Alleles
*Mutation
Myocardium/metabolism
Female
CRISPR-Cas Systems
Male
Heterozygote

@article {pmid40003596,
year = {2025},
author = {Rubí-Rangel, LM and León-Félix, J and Villicaña, C},
title = {Exploring Viral Interactions in Clavibacter Species: In Silico Analysis of Prophage Prevalence and Antiviral Defenses.},
journal = {Life (Basel, Switzerland)},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/life15020187},
pmid = {40003596},
issn = {2075-1729},
support = {P00768014//CIAD Internal Project/ ; 784//Investigadores por México CONAHCYT/ ; },
abstract = {Clavibacter is a phytopathogenic genus that causes severe diseases in economically important crops, yet the role of prophages in its evolution, pathogenicity, and adaptation remains poorly understood. In this study, we used PHASTER, Prophage Hunter, and VirSorter2 to identify prophage-like sequences in publicly available Clavibacter genomes. Prophage predictions were checked by hand to make them more accurate. We identified 353 prophages, predominantly in chromosomes, with some detected phage-plasmids. Most prophages exhibited traits of advanced domestication, such as an unimodal genome length distribution, reduced numbers of integrases, and minimal transposable elements, suggesting long-term interactions with their bacterial hosts. Comparative genomic analyses uncovered high genetic diversity, with distinct prophage clusters showing species-specific and interspecies conservation patterns. Functional annotation revealed prophage-encoded genes were involved in sugar metabolism, heavy metal resistance, virulence factors, and antibiotic resistance, highlighting their contribution to host fitness and environmental adaptation. Defense system analyses revealed that, despite lacking CRISPR-Cas, Clavibacter genomes harbor diverse antiviral systems, including PD-Lambda-1, AbiE, and MMB_gp29_gp30, some encoded within prophages. These findings underscore the pervasive presence of prophages in Clavibacter and their role in shaping bacterial adaptability and evolution.},
}

@article {pmid40003070,
year = {2025},
author = {Zhao, Z and Zou, X and Zhu, Y and He, Y and Jebessa, E and Zhang, J and Ji, J and Chen, P and Luo, C},
title = {Achieving Optimal Transfection Conditions in Chicken Primordial Germ Cells Under Feeder- and Serum-Free Medium.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {4},
pages = {},
doi = {10.3390/ani15040590},
pmid = {40003070},
issn = {2076-2615},
support = {2022B0202110002//the Science and Technology Program of Guangdong/ ; XT202217//the Project of Collaborative Innovation Center of GDAAS/ ; ZX202401-03//the sub project of "Discipline Construction of Swine and Poultry Breeding Industry" of Special Project on Science and Technology Innovation Strategy/ ; CARS-41//the earmarked fund/ ; GDNKY-ZQQZ-K8, GDNKY-ZQQZ-K11//the Project of State Key Laboratory of Swine and Poultry Breeding Industry/ ; },
abstract = {The successful application of primordial germ cells (PGCs) is an ideal method for generating gene-edited birds. However, barriers to efficient DNA transfection in PGCs lead to low transfection efficiency, limiting the generation of genetically modified chickens. The current study utilized chemical transfection and electroporation methods to determine the optimal transfection conditions for the PGC line under feeder- and serum-free medium. Among the tested methods, the Lonza electroporation system exhibited the highest transduction efficiency, with a previously unreported rate of 71.13 ± 1.26%. Optimal transfection conditions were achieved using 4 µg of DNA and 100 µL of Entranster[TM]-E in 1 × 10[6] PGCs. Furthermore, the optimal electroporation conditions resulted in low cell death and normal expression of pluripotency-related genes, highlighting the low cytotoxicity. The resulting electroporation models were then used to deliver the enhanced green fluorescent protein (EGFP) gene to the Z chromosome with a Cas9-gRNA plasmid, achieving a 7-day insertion efficiency of 14.63 ± 1.07%. Our study highlights the vast potential of electroporation technology for the transfection of PGCs.},
}

@article {pmid40001389,
year = {2025},
author = {Pandova, M and Kizheva, Y and Hristova, P},
title = {Relationship Between CRISPR-Cas Systems and Acquisition of Tetracycline Resistance in Non-Clinical Enterococcus Populations in Bulgaria.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {2},
pages = {},
doi = {10.3390/antibiotics14020145},
pmid = {40001389},
issn = {2079-6382},
support = {project No BG-RRP-2.004-0008//European Union-NextGenerationEU, National Recovery and Resilience Plan of the Republic of Bulgaria, SUMMIT 3.2.4./ ; },
abstract = {Non-clinical enterococci are relatively poorly studied by means of acquired antibiotic resistance to tetracycline and by the distribution, functionality and role of their CRISPR systems. Background: In our study, 72 enterococcal strains, isolated from various non-clinical origins, were investigated for their phenotypic and genotypic (tet(M), tet(O), tet(S), tet(L), tet(K), tet(T) and tet(W)) tetracycline resistance. Methods: The genetic determinants for HGT (MGEs (Int-Tn and prgW), inducible pheromones (cpd, cop and cff), aggregation substances (agg, asa1, prgB and asa373) and CRISPR-Cas systems were characterized by PCR and whole-genome sequencing. Results: Four tet genes (tetM, tetO, tetS and tetT) were detected in 39% (n = 28) of our enterococcal population, with tetM (31%) being dominant. The gene location was linked to the Tn6009 transposon. All strains that contained tet genes also had genes for HGT. No tet genes were found in E. casseliflavus and E. gilvus. In our study, 79% of all tet-positive strains correlated with non-functional CRISPR systems. The strain E. faecalis BM15 was the only one containing a combination of a functional CRISPR system (cas1, cas2, csn2 and csn1/cas9) and tet genes. The CRISPR subtype repeats II-A, III-B, IV-A2 and VI-B1 were identified among E. faecalis strains (CM4-II-A, III-B and VI-B1; BM5-IV-A2, II-A and III-B; BM12 and BM15-II-A). The subtype II-A was the most present. These repeats enclosed a great number of spacers (1-10 spacers) with lengths of 31 to 36 bp. One CRISPR locus was identified in plasmid (p.Firmicutes1 in strain E. faecalis BM5). We described the presence of CRISPR loci in the species E. pseudoavium, E. pallens and E. devriesei and their lack in E. gilvus, E. malodoratus and E. mundtii. Conclusions: Our findings generally describe the acquisition of foreign DNA as a consequence of CRISPR inactivation, and self-targeting spacers as the main cause.},
}

@article {pmid40001243,
year = {2025},
author = {Liu, Z and Li, Y and Wang, S and Wang, Y and Sui, M and Liu, J and Chen, P and Wang, J and Zhang, Y and Dang, C and Hou, P},
title = {Genome-wide CRISPR screening identifies PHF8 as an effective therapeutic target for KRAS- or BRAF-mutant colorectal cancers.},
journal = {Journal of experimental & clinical cancer research : CR},
volume = {44},
number = {1},
pages = {70},
pmid = {40001243},
issn = {1756-9966},
support = {82172675，82273036//National Natural Science Foundation of China/ ; 82303731//National Natural Science Foundation of China/ ; No. S2024-JC-QN-0868//Natural Science Basic Research Program of Shaanxi Province/ ; 2024JC-YBQN-0934//Natural Science Basic Research Program of Shaanxi Province/ ; 2022JQ-782//Natural Science Basic Research Program of Shaanxi Province/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology/drug therapy/metabolism ; *Proto-Oncogene Proteins B-raf/genetics/metabolism ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Mice ; Animals ; Mutation ; Cell Line, Tumor ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Gene Expression Regulation, Neoplastic ; Histone Demethylases ; },
abstract = {BACKGROUND: Mutations in KRAS and BRAF genes are prevalent in colorectal cancer (CRC), which strikingly promote tumorigenesis and lead to poor response to a variety of treatments including immunotherapy by activating the MAPK/ERK pathway. Thus, there is an urgent need to discover effective therapeutic targets and strategies.

METHODS: CRISPR-Cas9 lentiviral knockout library was used to screen the suppressors of anti-PD1 immunotherapy. Bioinformatic analysis was used to analyze the correlation between PHF8 expression and immune indicators in CRC. In vitro and in vivo experiments were utilized to determine the effects of PHF8 on the immune indexes and malignant phenotypes of CRC cells. qRT-PCR, western blotting, immunohistochemical (IHC) staining, and chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the regulatory effects of PHF8 on PD-L1, KRAS, BRAF, and c-Myc and the regulatory effect c-Myc/miR-22-3p signaling axis on PHF8 expression in CRC cells.

RESULTS: This study identified histone lysine demethylase PHF8 as a negative regulator for the efficacy of anti-PD1 therapy and found that it was highly expressed in CRCs and strongly associated with poor patient survival. Functional studies showed that PHF8 played an oncogenic role in KRAS- or BRAF-mutant CRC cells, but not in wild-type ones. Mechanistically, PHF8 up-regulated the expression of PD-L1, KRAS, BRAF, and c-Myc by increasing the levels of transcriptional activation marks H3K4me3 and H3K27ac and decreasing the levels of transcriptional repression mark H3K9me2 within their promoter regions, promoting immune escape and tumor progression. Besides, our data also demonstrated that PHF8 was up-regulated by the c-Myc/miR-22-3p signaling axis to form a positive feedback loop. Targeting PHF8 substantially improved the efficacy of anti-PD1 therapy and inhibited the malignant phenotypes of KRAS- or BRAF-mutant CRC cells.

CONCLUSION: Our data demonstrate that PHF8 may be an effective therapeutic target for KRAS- or BRAF-mutant CRCs.},
}

Humans
*Colorectal Neoplasms/genetics/pathology/drug therapy/metabolism
*Proto-Oncogene Proteins B-raf/genetics/metabolism
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*Transcription Factors/genetics/metabolism
Mice
Animals
Mutation
Cell Line, Tumor
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Female
Gene Expression Regulation, Neoplastic
Histone Demethylases

@article {pmid40001184,
year = {2025},
author = {Nikolouli, K and Compton, A and Tu, ZJ and Bourtzis, K},
title = {Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {76},
pmid = {40001184},
issn = {1756-3305},
support = {AI123338//National Institute of Allergy and Infectious Diseases/ ; AI123338//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Aedes/genetics/growth & development/physiology ; Animals ; Male ; Female ; *Mosquito Control/methods ; Genetic Markers ; Insect Proteins/genetics/metabolism ; Fertility/genetics ; Mosquito Vectors/genetics/growth & development ; Gene Knockout Techniques ; Mutation ; CRISPR-Cas Systems ; Genotype ; Phenotype ; Sex Ratio ; },
abstract = {BACKGROUND: Aedes aegypti is expected to invade previously unoccupied areas, mainly due to the climate change, the increase in travel and trade activities and the continuous transformation of the rural environment into urban areas. The sterile insect technique (SIT), which relies on the mass production and release of sterile males, is an environmentally friendly approach that can be applied for population control of Ae. aegypti. SIT programs can be greatly benefited by a genetic sexing strain (GSS) and a reliable sex sorting system to minimize any accidental female release. Visually detectable or conditionally lethal selectable markers can be used for the development of new GSSs. In this study, we evaluated the suitability and competence of a mutant Ae. aegypti ebony strain for the development of a new GSS. The ebony gene is known to be involved in the pigmentation pathway of several dipteran insects, including Ae. aegypti.

METHODS: An ebony gene knockout was developed though CRISPR/Cas9 mutagenesis. G0 individuals with the desired phenotype were crossed, and progeny were screened in every generation. PCR and sequencing were performed using gDNA from a pulled leg to determine the mutant genotype. Quality control tests, including pupae and adult recovery rates, male sex ratio and fecundity, were applied to the ebony mutant line to determine whether the mutation confers any fitness cost.

RESULTS: An Ae. aegypti ebony knockout mutant carrying a 5-bp deletion was obtained, which presented darker head and siphon phenotypes at the larval stage. However, genetic analysis revealed that this ebony mutation results in incomplete penetrance and variable expressivity. The establishment of a pure ebony mutant line was not possible because of the fitness costs conferred by the mutation.

CONCLUSIONS: In this study, the adequacy and suitability of the ebony gene as a selectable marker for the development of a GSS in Ae. aegypti were assessed. Despite its clear phenotype early in larval development, the homozygous mutant line presented phenotypic inconsistency and loss of fertility. These drawbacks clearly indicate that this particular mutation is not suitable for the development of a new GSS. Nonetheless, it cannot be excluded that a different mutation will lead to a different expression and penetrance profile and a viable homozygous mutant line.},
}

*Aedes/genetics/growth & development/physiology
Animals
Male
Female
*Mosquito Control/methods
Genetic Markers
Insect Proteins/genetics/metabolism
Fertility/genetics
Mosquito Vectors/genetics/growth & development
Gene Knockout Techniques
Mutation
CRISPR-Cas Systems
Genotype
Phenotype
Sex Ratio

@article {pmid40000946,
year = {2025},
author = {Rai, A and Skårn, MN and Elameen, A and Tengs, T and Amundsen, MR and Bjorå, OS and Haugland, LK and Yakovlev, IA and Brurberg, MB and Thorstensen, T},
title = {CRISPR-Cas9-mediated deletions of FvMYB46 in Fragaria vesca reveal its role in regulation of fruit set and phenylpropanoid biosynthesis.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {256},
pmid = {40000946},
issn = {1471-2229},
mesh = {*Fruit/genetics/growth & development/metabolism ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Fragaria/genetics/growth & development/metabolism ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; Flowers/genetics/growth & development/metabolism ; Flavonoids/biosynthesis/metabolism ; Lignin/biosynthesis/metabolism ; },
abstract = {The phenylpropanoid pathway, regulated by transcription factors of the MYB family, produces secondary metabolites that play important roles in fertilization and early phase of fruit development. The MYB46 transcription factor is a key regulator of secondary cell wall structure, lignin and flavonoid biosynthesis in many plants, but little is known about its activity in flowers and berries in F. vesca. For functional analysis of FvMYB46, we designed a CRISPR-Cas9 construct with an endogenous F. vesca-specific U6 promoter for efficient and specific expression of two gRNAs targeting the first exon of FvMYB46. This generated mutants with an in-frame 81-bp deletion of the first conserved MYB domain or an out-of-frame 82-bp deletion potentially knocking out gene function. In both types of mutant plants, pollen germination and fruit set were significantly reduced compared to wild type. Transcriptomic analysis of flowers revealed that FvMYB46 positively regulates the expression of genes involved in processes like xylan biosynthesis and metabolism, homeostasis of reactive oxygen species (ROS) and the phenylpropanoid pathway, including secondary cell wall biosynthesis and flavonoid biosynthesis. Genes regulating carbohydrate metabolism and signalling were also deregulated, suggesting that FvMYB46 might regulate the crosstalk between carbohydrate metabolism and phenylpropanoid biosynthesis. In the FvMYB46-mutant flowers, the flavanol and flavan-3-ol contents, especially epicatechin, quercetin-glucoside and kaempferol-3-coumaroylhexoside, were reduced, and we observed a local reduction in the lignin content in the anthers. Together, these results suggest that FvMYB46 controls fertility and efficient fruit set by regulating the cell wall structure, flavonoid biosynthesis, carbohydrate metabolism, and sugar and ROS signalling in flowers and early fruit development in F. vesca.},
}

*Fruit/genetics/growth & development/metabolism
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
*Fragaria/genetics/growth & development/metabolism
*Transcription Factors/genetics/metabolism
Gene Expression Regulation, Plant
Flowers/genetics/growth & development/metabolism
Flavonoids/biosynthesis/metabolism
Lignin/biosynthesis/metabolism

@article {pmid40000476,
year = {2025},
author = {Sun, W and Ren, X and Xiao, Y and Li, B and Pang, QA and Yang, M and Zhu, R and Guo, Z and Yu, J and Huang, J and Wang, Y and Liu, S},
title = {Fluorescent/colorimetric dual-mode for detecting of MC-LR using bidirectional RCA coupled with CdTe QDs.},
journal = {Mikrochimica acta},
volume = {192},
number = {3},
pages = {189},
pmid = {40000476},
issn = {1436-5073},
support = {ts201712048//Program for Taishan Scholar of Shandong Province/ ; 32072330//National Natural Science Foundation of China/ ; ZR2022MB066//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Tellurium/chemistry ; *Quantum Dots/chemistry ; *Cadmium Compounds/chemistry ; *Microcystins/analysis ; *Colorimetry/methods ; *Biosensing Techniques/methods ; *Marine Toxins/analysis ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Spectrometry, Fluorescence/methods ; CRISPR-Cas Systems ; Fluorescent Dyes/chemistry ; },
abstract = {A fluorescent/colorimetric dual-mode biosensor was designed using CdTe QDs and CRISPR/Cas for the efficient and ultrasensitive detection of microcystin-leucine-arginine (MC-LR). The biosensor mainly activates the trans-cleavage activity of Cas12a through nucleic acid amplification technology, such as bidirectional rolling circle amplification (B-RCA), to perform signal conversion, release Ag[+] from the cleaving hairpin, quench QD fluorescence, and perform signal presentation. The biosensor can perform fluorometric and colorimetric detection, enabling rapid field assays. It exhibits enhanced selectivity, increased sensitivity, and greater accuracy. The optimal conditions yield a detection range from 0.05 to 500 nM, with a minimum detectable concentration of 2.137 pM, surpassing the performance of traditional methods. The biosensor can effectively detect MC-LR in actual environmental samples. Overall, this study provided a general detection approach for the application of nucleic acid detection technology to detect trace pollutants in the environment.},
}

*Tellurium/chemistry
*Quantum Dots/chemistry
*Cadmium Compounds/chemistry
*Microcystins/analysis
*Colorimetry/methods
*Biosensing Techniques/methods
*Marine Toxins/analysis
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Spectrometry, Fluorescence/methods
CRISPR-Cas Systems
Fluorescent Dyes/chemistry

@article {pmid39999982,
year = {2025},
author = {Chia, BS and Seah, YFS and Wang, B and Shen, K and Srivastava, D and Chew, WL},
title = {Engineering a New Generation of Gene Editors: Integrating Synthetic Biology and AI Innovations.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.4c00686},
pmid = {39999982},
issn = {2161-5063},
abstract = {CRISPR-Cas technology has revolutionized biology by enabling precise DNA and RNA edits with ease. However, significant challenges remain for translating this technology into clinical applications. Traditional protein engineering methods, such as rational design, mutagenesis screens, and directed evolution, have been used to address issues like low efficacy, specificity, and high immunogenicity. These methods are labor-intensive, time-consuming, and resource-intensive and often require detailed structural knowledge. Recently, computational strategies have emerged as powerful solutions to these limitations. Using artificial intelligence (AI) and machine learning (ML), the discovery and design of novel gene-editing enzymes can be streamlined. AI/ML models predict activity, specificity, and immunogenicity while also enhancing mutagenesis screens and directed evolution. These approaches not only accelerate rational design but also create new opportunities for developing safer and more efficient genome-editing tools, which could eventually be translated into the clinic.},
}

@article {pmid39999874,
year = {2025},
author = {Carbajo, CG and Han, X and Savur, B and Upadhyaya, A and Taha, F and Tinti, M and Wheeler, RJ and Yates, PA and Tiengwe, C},
title = {A high-throughput protein tagging toolkit that retains endogenous untranslated regions for studying gene regulation in kinetoplastids.},
journal = {Open biology},
volume = {15},
number = {2},
pages = {240334},
doi = {10.1098/rsob.240334},
pmid = {39999874},
issn = {2046-2441},
support = {/WT_/Wellcome Trust/United Kingdom ; /HI/NHLBI NIH HHS/United States ; },
mesh = {*Protozoan Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Expression Regulation ; *Trypanosoma brucei brucei/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Kinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology. We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide from Thosea asigna to generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events. We validate the system by tagging six Trypanosoma brucei proteins and demonstrate (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.},
}

*Protozoan Proteins/genetics/metabolism
*CRISPR-Cas Systems
*Gene Expression Regulation
*Trypanosoma brucei brucei/genetics/metabolism
Gene Editing/methods

@article {pmid39999224,
year = {2025},
author = {Gertsenstein, M and Lintott, LG and Nutter, LMJ},
title = {Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease.},
journal = {Current protocols},
volume = {5},
number = {2},
pages = {e70109},
pmid = {39999224},
issn = {2691-1299},
mesh = {Animals ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Alleles ; *Epitopes/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Microinjections ; Electroporation ; Zygote/metabolism ; Endonucleases/genetics/metabolism ; },
abstract = {Mice carrying patient-associated base changes are powerful tools to define the causality of single-nucleotide variants to disease states. Epitope tags enable immuno-based studies of genes for which no antibodies are available. These alleles enable detailed and precise developmental, mechanistic, and translational research. The first step in generating these alleles is to identify within the target sequence-the orthologous sequence for base changes or the N or C terminus for epitope tags-appropriate Cas9 protospacer sequences. Subsequent steps include design and acquisition of a single-stranded oligonucleotide repair template, synthesis of a single guide RNA (sgRNA), collection of zygotes, and microinjection or electroporation of zygotes with Cas9 mRNA or protein, sgRNA, and repair template followed by screening born mice for the presence of the desired sequence change. Quality control of mouse lines includes screening for random or multicopy insertions of the repair template and, depending on sgRNA sequence, off-target sequence variation introduced by Cas9. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Single guide RNA design and synthesis Alternate Protocol 1: Single guide RNA synthesis by primer extension and in vitro transcription Basic Protocol 2: Design of oligonucleotide repair template Basic Protocol 3: Preparation of RNA mixture for microinjection Support Protocol 1: Preparation of microinjection buffer Alternate Protocol 2: Preparation of RNP complexes for electroporation Basic Protocol 4: Collection and preparation of mouse zygotes for microinjection or electroporation Basic Protocol 5: Electroporation of Cas9 RNP into zygotes using cuvettes Alternate Protocol 3: Electroporation of Cas9 RNP into zygotes using electrode slides Basic Protocol 6: Screening and quality control of derived mice Support Protocol 2: Deconvoluting multiple sequence chromatograms with DECODR.},
}

Animals
Mice
*RNA, Guide, CRISPR-Cas Systems/genetics
*Alleles
*Epitopes/genetics
Gene Editing/methods
CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism
Microinjections
Electroporation
Zygote/metabolism
Endonucleases/genetics/metabolism

@article {pmid39585742,
year = {2025},
author = {Tan, YY and Liew, YY and Lee, RRQ and Castel, B and Chan, NM and Wan, WL and Zhang, Y and Hu, D and Chan, P and Kim, ST and Chae, E},
title = {Generation of Inheritable A-to-G Transitions Using Adenine Base Editing and NG-PAM Cas9 in Arabidopsis thaliana.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {38},
number = {1},
pages = {30-42},
doi = {10.1094/MPMI-10-24-0127-TA},
pmid = {39585742},
issn = {0894-0282},
mesh = {*Arabidopsis/genetics ; *Gene Editing ; *Adenine/metabolism ; *CRISPR-Cas Systems ; Plants, Genetically Modified ; Arabidopsis Proteins/genetics/metabolism ; Adenosine Deaminase/metabolism/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {Towards precise genome editing, base editors have been developed by fusing catalytically compromised Cas9 with deaminase components, mediating C-to-T (cytosine base editors) or A-to-G (adenine base editors) transition. We developed a set of vectors consisting of a 5'-NG-3' PAM-recognizing variant of SpCas9 with adenosine deaminases TadA7.10 or TadA8e. Using a phenotype-based screen in Arabidopsis thaliana targeting multiple PDS3 intron splice sites, we achieved up to 81% somatic A-to-G editing in primary transformants at a splice acceptor site with NGG PAM, while 35% was achieved for the same target adenine with NGA PAM. Among tested vectors, pECNUS4 (Addgene #184887), carrying TadA8e, showed the highest adenine base editor (ABE) efficiency. With pECNUS4, we recreated a naturally occurring allele of DANGEROUS MIX3 (DM3) in two generations, transgene-free, for NGC PAM. We also simultaneously base-edited four redundant DM1/SSI4 homologs, encoding nucleotide-binding leucine-rich repeat (NLR) proteins, using a single gRNA with NGA PAM targeting the conserved yet functionally crucial P-loop motif of NLR proteins. We found fixation of A-to-G in three NLR genes for all three possible adenine sites within base-editing window 3-9, as the edited genes segregate in T2. Multigene targeting succeeded in rescuing the previously reported autoimmune phenotype in two generations. Mediating desired ABE on seven NLR genes simultaneously was successful as well; above 77% editing was achieved in six of the seven possible targets in a T1 plant, with the remaining having a moderately high (32%) editing. ABE application to specifically inactivate functional motifs is anticipated to expedite the discovery of novel roles for proteins. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.},
}

*Arabidopsis/genetics
*Gene Editing
*Adenine/metabolism
*CRISPR-Cas Systems
Plants, Genetically Modified
Arabidopsis Proteins/genetics/metabolism
Adenosine Deaminase/metabolism/genetics
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39998577,
year = {2025},
author = {Liu, J and Wang, Y and Zhang, X and Huang, M and Li, G},
title = {Direct lysis combined with amplification-free CRISPR/Cas12a-SERS genosensor for ultrafast and on-site identification of meat authenticity.},
journal = {Mikrochimica acta},
volume = {192},
number = {3},
pages = {187},
pmid = {39998577},
issn = {1436-5073},
mesh = {*CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Food Contamination/analysis ; *Meat/analysis ; Limit of Detection ; Animals ; Polystyrenes/chemistry ; Microspheres ; Biosensing Techniques/methods ; DNA, Single-Stranded/chemistry/genetics ; Bacterial Proteins/genetics ; Nitriles/chemistry ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {A novel direct lysis method combined with amplification-free CRISPR/Cas12a-SERS genosensor was for the first time developed to rapidly and sensitively identify meat adulteration. Notably, polystyrene (PS) microspheres, with distinct shrinking and swelling properties, were dexterously employed to encapsulate biological-silent Raman reporter 4-mercaptobenzonitrile (4-MBN) and act as a controlled-release signal probe. Target DNA activated the trans-cleavage activity of CRISPR/Cas12a towards ssDNA linked with PS microsphere to liberate the signal probe, which was able to release numerous Raman reporters after treatment with THF solution, resulting in high signal amplification. Through this platform, trace target DNA was deftly transformed into a sensitive Raman signal and could be on-site determined through a portable Raman equipment. Under optimized conditions, this strategy displayed good linearity in the range 1-450 ng/μL (R[2] = 0.9943) and favorable sensitivity with limit of detection as low as 0.23 ng/μL without any pre-amplification. Moreover, it exhibited good applicability to on-site identification of commercial meat samples in complicated food matrix. In addition, DNA extraction by direct lysis and amplification-free detection realized ultrafast meat adulteration determination within 35 min from sampling to result. This method possessed great potential in rapid and on-site accurate determination of meat authenticity.},
}

*CRISPR-Cas Systems/genetics
*Spectrum Analysis, Raman/methods
*Food Contamination/analysis
*Meat/analysis
Limit of Detection
Animals
Polystyrenes/chemistry
Microspheres
Biosensing Techniques/methods
DNA, Single-Stranded/chemistry/genetics
Bacterial Proteins/genetics
Nitriles/chemistry
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39998178,
year = {2025},
author = {van den Berg, DF and Brouns, SJJ},
title = {Reduced prevalence of phage defense systems in Pseudomonas aeruginosa strains from cystic fibrosis patients.},
journal = {mBio},
volume = {},
number = {},
pages = {e0354824},
doi = {10.1128/mbio.03548-24},
pmid = {39998178},
issn = {2150-7511},
abstract = {Cystic fibrosis is a genetic disorder that affects mucus clearance, particularly of the lungs. As a result, cystic fibrosis patients often experience infections from bacteria, which contribute to the disease progression. Pseudomonas aeruginosa is one of the most common opportunistic pathogens associated with cystic fibrosis. The presence of P. aeruginosa complicates the treatment due to its high antibiotic resistance. Thus, research is ongoing to treat these infections with bacterial viruses instead, known as bacteriophages. Notably, P. aeruginosa clinical strains possess a variety of phage defense mechanisms that may limit the effectiveness of phage therapy. In this study, we compared the defense system repertoire of P. aeruginosa strains isolated from cystic fibrosis patients with those from non-cystic fibrosis patients. Our findings reveal that P. aeruginosa strains isolated from cystic fibrosis patients have fewer phage defense mechanisms per strain than from non-cystic fibrosis patients, suggesting altered phage selection pressures in strains colonizing CF patient lungs.IMPORTANCECystic fibrosis patients often experience chronic Pseudomonas aeruginosa lung infections, which are challenging to treat with antibiotics and contribute to disease progression and eventual respiratory failure. Phage therapy is being explored as an alternative treatment strategy for these infections. However, assessing strain susceptibility to phage treatment is essential for ensuring efficacy. To address this, we investigated whether CF-associated clinical P. aeruginosa strains have a distinct phage defense repertoire compared with those isolated from other lung patients. We observed that CF-associated P. aeruginosa strains have significantly fewer phage defenses, possibly affecting the susceptibility of these strains to phage infection.},
}

@article {pmid39997222,
year = {2025},
author = {Hu, R and Guo, C and Liu, X and Lin, Y and Yang, Z and Li, Z and Yang, Y and Ma, E and Li, Y and Chen, J and Liu, L},
title = {Structural basis of ssDNA-guided NADase activation of prokaryotic SPARTA system.},
journal = {Nucleic acids research},
volume = {53},
number = {4},
pages = {},
doi = {10.1093/nar/gkaf110},
pmid = {39997222},
issn = {1362-4962},
support = {32171286//National Natural Science Foundation of China/ ; 3502Z20227020//Natural Science Foundation of Xiamen, China/ ; 2024J011007//Natural Science Foundation of Fujian Province/ ; },
mesh = {*DNA, Single-Stranded/metabolism/chemistry ; Models, Molecular ; Crystallography, X-Ray ; Argonaute Proteins/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Enzyme Activation ; NAD/metabolism ; Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; },
abstract = {Short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins constitute a prokaryotic immune system, mediating RNA- or DNA-guided target single-stranded DNA (ssDNA) to activate NADase activity and induce cell death by degrading NAD+ in response to invading plasmids. Although the guide RNA-mediated targeting mechanism of SPARTA has been established, the functional role and mechanisms of guide DNA-mediated SPARTA remain poorly understood. Here, we report two crystal structures of Crenotalea thermophila SPARTA complexes with 5'-phosphorylated 21-nt guide DNA and complementary target ssDNA lengths of 15 or 20 nt. The structures demonstrate specific recognition of the 5'-OH or 3'-OH groups in target DNA by SPARTA, while not recognizing the 5'-P group in guide DNA. This suggests distinct recognition models for guide DNA and guide RNA, indicating different activation mechanisms. Furthermore, these two structures reveal disparate models for recognizing guide DNA and target DNA, providing insights into the length requirement for SPARTA activation.},
}

*DNA, Single-Stranded/metabolism/chemistry
Models, Molecular
Crystallography, X-Ray
Argonaute Proteins/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
Enzyme Activation
NAD/metabolism
Bacterial Proteins/chemistry/metabolism/genetics
Protein Binding

@article {pmid39996750,
year = {2025},
author = {Mentani, A and Maresca, M and Shiriaeva, A},
title = {Prime Editing: Mechanistic Insights and DNA Repair Modulation.},
journal = {Cells},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/cells14040277},
pmid = {39996750},
issn = {2073-4409},
support = {101072427//European Union's Horizon 2021/ ; },
mesh = {Humans ; *Gene Editing/methods ; *DNA Repair ; Animals ; CRISPR-Cas Systems/genetics ; DNA/metabolism/genetics ; },
abstract = {Prime editing is a genome editing technique that allows precise modifications of cellular DNA without relying on donor DNA templates. Recently, several different prime editor proteins have been published in the literature, relying on single- or double-strand breaks. When prime editing occurs, the DNA undergoes one of several DNA repair pathways, and these processes can be modulated with the use of inhibitors. Firstly, this review provides an overview of several DNA repair mechanisms and their modulation by known inhibitors. In addition, we summarize different published prime editors and provide a comprehensive overview of associated DNA repair mechanisms. Finally, we discuss the delivery and safety aspects of prime editing.},
}

Humans
*Gene Editing/methods
*DNA Repair
Animals
CRISPR-Cas Systems/genetics
DNA/metabolism/genetics

@article {pmid39996452,
year = {2025},
author = {Tang, H and Xu, F and Sun, D and Hua, L and Xiong, J and Xu, M and Xu, J and Zhong, P},
title = {Cas9 Mouse Model of Skull Base Meningioma Driven by Combinational Gene Inactivation in Meningeal Cells.},
journal = {CNS neuroscience & therapeutics},
volume = {31},
number = {2},
pages = {e70287},
doi = {10.1111/cns.70287},
pmid = {39996452},
issn = {1755-5949},
mesh = {Animals ; *Meningioma/genetics/pathology ; *Mice, Transgenic ; Mice ; *Disease Models, Animal ; *Meningeal Neoplasms/genetics/pathology ; Skull Base Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Mice, Inbred C57BL ; Lentivirus/genetics ; },
abstract = {INTRODUCTION: Neurofibromatosis type 2 (Nf2) gene inactivation is common in sporadic and Nf2-related meningioma. There is currently scant literature describing the development of an intracranial meningioma model in animals. Given the role of Nf2 and other gene inactivation in meningeal cells, we used Cas9 mice here as the background host to establish a new animal model of skull base meningioma in this study.

AIMS: Cas9 transgenic mice were purchased from Jackson Laboratory and raised in our institution. Subsequently, meningeal cells were obtained from the Cas9 transgenic mice, cultured in medium, and passaged in vitro. We then prepared lentivirus vector pLentiCre/gRNA, which could express the elements blocking the function of four genes: Nf2, P15[Ink4b], P16[Ink4a], and P19[Arf]. We infected the meningeal cells with the lentivirus vector pLentiCre/gRNA and tested the expression of these four genes in those infected meningeal cells. Next, adeno-associated virus vector pAAVCre/gRNA was injected in vivo into the skull base meningeal cells of the neonate Cas9 transgenic mice. These mice were observed once a week and killed 10 months later for brain inspection and pathological analysis.

RESULTS: Twenty Cas9 transgenic mice were successfully bred. Five mice were killed so that meningeal cells could be extracted, cultured, and infected with the lentivirus vector pLentiCre/gRNA for 72 h in vitro. The gene function test showed that Nf2, P15[Ink4b], P16[Ink4a], and P19[Arf] were all blocked in the infected meningeal cells, which indicated that the lentivirus vector pLentiCre/gRNA could effectively block the expression of the four genes in targeted cells. Then pAAVCre/gRNA was injected into the skull base meningeal cells of 15 mice in vivo, and nine mice were observed for 10 months so that the intracranial tumor growth could be assessed. Among these nine mice, pathological analysis showed that six mice had benign meningioma subtypes similar to human meningioma, one mouse had atypical meningioma, one mouse had malignant meningioma, and one mouse had sarcoma.

CONCLUSIONS: The Cas9 mouse model of skull base meningioma generated with the Nf2 genetic defect and the combinational loss of P15[Ink4b], P16[Ink4a], and P19[Arf] could provide a new tool for investigating the pathogenesis of meningioma and the development of chemical interventions for this disease.},
}

Animals
*Meningioma/genetics/pathology
*Mice, Transgenic
Mice
*Disease Models, Animal
*Meningeal Neoplasms/genetics/pathology
Skull Base Neoplasms/genetics/pathology
CRISPR-Cas Systems
Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
Mice, Inbred C57BL
Lentivirus/genetics

@article {pmid39995605,
year = {2025},
author = {Wang, D and Mandal, P and Rahman, MS and Yang, L},
title = {Engineering tomato disease resistance by manipulating susceptibility genes.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1537148},
pmid = {39995605},
issn = {2673-3439},
abstract = {Various pathogens severely threaten tomato yield and quality. Advances in understanding plant-pathogen interactions have revealed the intricate roles of resistance (R) and susceptibility (S) genes in determining plant immunity. While R genes provide targeted pathogen resistance, they are often vulnerable to pathogen evolution. Conversely, S genes offer a promising avenue for developing broad-spectrum and durable resistance through targeted gene editing. Recent breakthroughs in CRISPR/Cas-based technologies have revolutionized the manipulation of plant genomes, enabling precise modification of S genes to enhance disease resistance in tomato without compromising growth or quality. However, the utilization of the full potential of this technique is challenging due to the complex plant-pathogen interactions and current technological limitations. This review highlights key advances in using gene editing tools to dissect and engineer tomato S genes for improved immunity. We discuss how S genes influence pathogen entry, immune suppression, and nutrient acquisition, and how their targeted editing has conferred resistance to bacterial, fungal, and viral pathogens. Furthermore, we address the challenges associated with growth-defense trade-offs and propose strategies, such as hormonal pathway modulation and precise regulatory edits, to overcome these limitations. This review underscores the potential of CRISPR-based approaches to transform tomato breeding, paving the way for sustainable production of disease-resistant cultivars amidst escalating global food security challenges.},
}

@article {pmid39995348,
year = {2025},
author = {Chen, Q and Sun, Y and Yao, J and Lu, Y and Qiu, R and Zhou, F and Deng, Z and Sun, Y},
title = {Engineering of Peptide-Inserted Base Editors with Enhanced Accuracy and Security.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e2411583},
doi = {10.1002/smll.202411583},
pmid = {39995348},
issn = {1613-6829},
support = {2018YFA0903200//National Key R&D Program of China/ ; 31920103001//Funds for International Cooperation and Exchange of the National Natural Science Foundation of China/ ; },
abstract = {Base editors are effective tools for introducing base conversions without double-strand breaks, showing broad applications in biotechnological and clinical areas. However, their non-negligible bystander mutations and off-target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific peptide fragments into the substrate binding pocket of deaminases in base editors to modify these outcomes. It is validated that the composition and position of the inserted peptide can significantly impact the performance of A3A-based cytosine base editor and TadA-8e-based adenine base editor, leading to improved editing activity and precision in human HEK293T cells. Importantly, the TadA-8e variant with DPLVLRRRQ peptide inserted behind S116 residue showed a strong motif preference of Y4A5N6, which can accurately edit the A5 base in targeted protospacer with minimized bystander and off-target effects in DNA and RNA-level. By summarizing the regularity during engineering, a set of systematic procedures is established, which can potentially be used to modify other types of base editors and make them more accurate and secure. In addition, the peptide insertion strategy is also proven to be compatible with traditional amino acid changes which have been reported, exhibiting excellent compatibility.},
}

@article {pmid39995104,
year = {2025},
author = {Bhatt, D and Sundaram, RK and López, KSL and Lee, T and Gueble, SE and Vasquez, JC},
title = {Development of Syngeneic Murine Glioma Models with Somatic Mismatch Repair Deficiency to Study Therapeutic Responses to Alkylating Agents and Immunotherapy.},
journal = {Current protocols},
volume = {5},
number = {2},
pages = {e70097},
doi = {10.1002/cpz1.70097},
pmid = {39995104},
issn = {2691-1299},
mesh = {Animals ; Mice ; *Glioma/drug therapy/genetics/immunology/pathology ; *Temozolomide/pharmacology/therapeutic use ; Cell Line, Tumor ; *Antineoplastic Agents, Alkylating/pharmacology/therapeutic use ; *Immunotherapy ; *DNA Mismatch Repair/drug effects ; *Disease Models, Animal ; Brain Neoplasms/drug therapy/genetics/immunology/pathology ; MutL Protein Homolog 1/genetics ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Neoplastic Syndromes, Hereditary/genetics/drug therapy ; },
abstract = {Glioblastoma (GBM) carries a dismal prognosis, with a median survival of less than 15 months. Temozolomide (TMZ), the standard frontline chemotherapeutic for GBM, is an alkylating agent that generates DNA O[6]-methylguanine (O[6]MeG) lesions. Without O[6]MeG-methyltransferase (MGMT), this lesion triggers the mismatch repair (MMR) pathway and leads to cytotoxicity via futile cycling. TMZ resistance frequently arises via the somatic acquisition of MMR deficiency (MMRd). Moreover, DNA-damaging agents have been shown capable of increasing tumor immunogenicity and improving response to immune checkpoint blockade (ICB), which has had limited success in glioma. The study of how alkylating chemotherapy such as TMZ impacts antitumor immunity in glioma has been hindered by a lack of immunocompetent models that incorporate relevant DNA repair genotypes. Here, we used CRISPR/Cas9 to generate models isogenic for knockout (KO) of Mlh1 in the syngeneic SB28 murine glioma cell line. MMR KO models readily formed intracranial tumors and exhibited in vitro and in vivo resistance to TMZ. In contrast, MMR KO cells maintained sensitivity to KL-50, a newly developed alkylating compound that exerts MGMT-dependent, MMR-independent cytotoxicity. Lastly, MMR KO tumors remained resistant to ICB, mirroring the lack of response seen in patients with somatic MMRd GBM. The development of syngeneic, immunologically cold glioma models with somatic loss of MMR will facilitate future studies on the immunomodulatory effects of alkylating agents in relevant DNA repair contexts, which will be vital for optimizing combinations with ICB. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Validation of mismatch repair knockouts and in vitro sensitivity to alkylating agents Basic Protocol 2: Stereotaxic injection of isogenic SB28 cells in female C57BL/6J mice and in vivo treatment.},
}

Animals
Mice
*Glioma/drug therapy/genetics/immunology/pathology
*Temozolomide/pharmacology/therapeutic use
Cell Line, Tumor
*Antineoplastic Agents, Alkylating/pharmacology/therapeutic use
*Immunotherapy
*DNA Mismatch Repair/drug effects
*Disease Models, Animal
Brain Neoplasms/drug therapy/genetics/immunology/pathology
MutL Protein Homolog 1/genetics
Mice, Inbred C57BL
CRISPR-Cas Systems
Neoplastic Syndromes, Hereditary/genetics/drug therapy

@article {pmid39995038,
year = {2025},
author = {Yin, N and Yu, H and Zhang, L and Luo, F and Wang, W and Han, X and He, Y and Zhang, Y and Wu, Y and Pu, J and Feng, T and Yang, G and Chen, T and Xie, G},
title = {Regulation of CRISPR trans-cleavage activity by an overhanging activator.},
journal = {Nucleic acids research},
volume = {53},
number = {4},
pages = {},
doi = {10.1093/nar/gkaf117},
pmid = {39995038},
issn = {1362-4962},
support = {2022YFC2603800//National Key R&D Program of China/ ; 82172369//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system exhibits extraordinary capability in the field of molecular diagnosis and biosensing, attributed to its trans-cleavage ability. The precise modulation of performance has emerged as a significant challenge in advancing CRISPR technology to the next stage of development. Herein, we reported a CRISPR/Cas12a regulation strategy based on an overhanging activator. The presence of overhanging domains in activators creates steric hindrances that have a substantial impact on the trans-cleavage activity and activation timing of Cas12a. The trans-cleavage activity of Cas12a can be finely tuned by adjusting the position, length, and complementarity of the overhanging domains. Moreover, specific structures exhibit characteristics of automatic delayed activation. The presence of overhanging domains enables precise and timely activation of Cas12a, facilitating multifunctional applications. This system effectively accomplishes dynamic regulation, programmable release of cargo, logical operations, and multi-enzyme detection. The flexibility and versatility of this simple and powerful CRISPR regulatory strategy will pave the way for expanded applications of CRISPR/Cas in biotechnology, bioengineering, and biomedicine.},
}

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

@article {pmid39994790,
year = {2025},
author = {Jiang, J and Cienfuegos-Gallet, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L},
title = {Correction: Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.},
journal = {Genome medicine},
volume = {17},
number = {1},
pages = {13},
pmid = {39994790},
issn = {1756-994X},
}

@article {pmid39994507,
year = {2025},
author = {Hossain, SM and Rao, Y and Hossain, JO and Pritchard, JR and Zhao, B},
title = {goloco: a web application to create genome scale information from surprisingly small experiments.},
journal = {BMC bioinformatics},
volume = {26},
number = {1},
pages = {61},
pmid = {39994507},
issn = {1471-2105},
mesh = {*Genomics/methods ; *Software ; *Internet ; Genome ; Phenotype ; CRISPR-Cas Systems/genetics ; Humans ; },
abstract = {BACKGROUND: Functional genomics aims to decipher gene function by observing cellular changes when specific genes are disrupted using CRISPR technology. However, these experiments are limited by scalability, as comprehensive CRISPR screens require extensive resources, involving millions of cells and thousands of sgRNAs, making large-scale studies challenging. We propose a novel approach with "CRISPR lossy compression" to reduce the complexity of CRISPR screens by focusing on key genetic nodes that can infer genome-wide phenotypes. These condensed sets, comprising 100 to 1,000 genes, enable previously impractical genome-wide screens tractable.

RESULTS: To make this approach accessible to the wider scientific community, we developed goloco, an interactive web application that allows users to explore genome-scale loss-of-function phenotypes from as few as 100 pooled measurements. The tool is complemented by a wide array of analyses, including volcano plot visualizations, regression and network analyses.

CONCLUSIONS: This tool goloco empowers researchers to conduct genome-scale functional studies with minimal experimental overhead, broadening the accessibility of large-scale functional genomics research.},
}

*Genomics/methods
*Software
*Internet
Genome
Phenotype
CRISPR-Cas Systems/genetics
Humans

@article {pmid39993805,
year = {2025},
author = {Weidle, UH and Birzele, F},
title = {Prostate Cancer: De-regulated Circular RNAs With Efficacy in Preclinical In Vivo Models.},
journal = {Cancer genomics & proteomics},
volume = {22},
number = {2},
pages = {136-165},
doi = {10.21873/cgp.20494},
pmid = {39993805},
issn = {1790-6245},
mesh = {Humans ; Male ; *Prostatic Neoplasms/genetics/pathology/metabolism/therapy ; *RNA, Circular/genetics ; Animals ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; },
abstract = {Therapy resistance, including castration-resistance and metastasis, remains a major hurdle in the treatment of prostate cancer. In order to identify novel therapeutic targets and treatment modalities for prostate cancer, we conducted a comprehensive literature search on PubMed to identify de-regulated circular RNAs that influence treatment efficacy in preclinical prostate cancer-related in vivo models. Our analysis identified 49 circular RNAs associated with various processes, including treatment resistance, transmembrane and secreted proteins, transcription factors, signaling cascades, human antigen R, nuclear receptor binding, ubiquitination, metabolism, epigenetics and other target categories. The identified targets and circular RNAs can be further scrutinized through target validation approaches. Down-regulated circular RNAs are candidates for reconstitution therapy, while up-regulated RNAs can be inhibited using small interfering RNA (siRNA), antisense oligonucleotides (ASO) or clustered regularly interspaced short palindromic repeats/CRISPR associated (CRISPR-CAS)-related approaches.},
}

Humans
Male
*Prostatic Neoplasms/genetics/pathology/metabolism/therapy
*RNA, Circular/genetics
Animals
Gene Expression Regulation, Neoplastic
Disease Models, Animal

@article {pmid39992915,
year = {2025},
author = {Gowen, BG and Melton, K and Leong, WI and Khekare, P and McCawley, S and Chan, J and Boivin, P and Jani, V and Cantor, AJ and Tambe, A and Haak-Frendscho, M and Janatpour, MJ and Wei, SC},
title = {Systematic identification and characterization of high efficiency Cas9 guide RNAs for therapeutic targeting of ADAR.},
journal = {PloS one},
volume = {20},
number = {2},
pages = {e0317745},
pmid = {39992915},
issn = {1932-6203},
mesh = {*Adenosine Deaminase/genetics/metabolism ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA-Binding Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {Therapeutic targeting of the adenosine deaminase ADAR has great potential in cancer and other indications; however, it remains unclear what approach can enable effective and selective therapeutic inhibition. Herein, we conduct multi-staged guide RNA screening and identify high efficiency Cas9 guide RNAs to enable a CRISPR/Cas-based approach for ADAR knockout. Through characterization in human primary immune cell systems we observe similar activity with two-part guide RNA and single guide RNA, dose responsive activity, similar guide activity rank order across different cell types, and favorable computational off-target profiles of candidate guide RNAs. We determine that knockout of ADAR using these guide RNAs induces pharmacodynamic responses primarily consisting of immunological responses such as a type I interferon response, consistent with the known function of ADAR as a key regulator of dsRNA sensing. We observe similar biological effects with targeting only the p150 isoform or both p110 and p150 isoforms of ADAR, indicating that at least in the contexts evaluated, loss of p150 ADAR mediates the primary response. These findings provide a resource of well-characterized, high efficiency ADAR-targeting Cas9 guide RNAs suitable for genomic medicines utilizing different delivery modalities and addressing different therapeutic areas.},
}

*Adenosine Deaminase/genetics/metabolism
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*RNA-Binding Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
Gene Knockout Techniques
HEK293 Cells
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid39992908,
year = {2025},
author = {Nemeth, T and Zarnocki, A and Ladanyi, A and Papp, C and Ayaydin, F and Szebeni, GJ and Gacser, A},
title = {PCR-based CRISPR/Cas9 system for fluorescent tagging: A tool for studying Candida parapsilosis virulence.},
journal = {PloS one},
volume = {20},
number = {2},
pages = {e0312948},
doi = {10.1371/journal.pone.0312948},
pmid = {39992908},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems ; Mice ; *Candida parapsilosis/genetics/pathogenicity ; Animals ; Virulence/genetics ; Polymerase Chain Reaction/methods ; Macrophages/microbiology ; Humans ; },
abstract = {Candida parapsilosis is persistent in a hospital environment hence it is often associated with nosocomial infections especially amongst low-birth weight neonates. Genetic modification is therefore important to characterise the physiological and virulence related properties of this fungus. A PCR-based CRISPR/Cas9 system has been adopted to facilitate the generation of fluorescent tagged prototroph isolates. We examined a total of eight fluorescent protein coding genes, out of which three were found to be applicable for simultaneous utilisation. We investigated three clinical isolates of C. parapsilosis in terms of their adherence to silicone and their uptake by J774.2 murine macrophages in competition assays. Interestingly, we found significant differences between them in both experiments where GA1 isolate was significantly less resistant to macrophage uptake and CDC317 was significantly more adherent to silicone material. In silico analysis of the agglutinin-like sequences (Als) exposed remarkable diversity in this protein family and additionally, the thorough analysis of the ALS genes revealed evidence of formation of a new gene by intrachromosomal recombination in the GA1 isolate. Finally, we provide a step by step protocol for the application of the PCR-based CRISPR/Cas9 system for fluorescently labelling C. parapsilosis isolates.},
}

*CRISPR-Cas Systems
Mice
*Candida parapsilosis/genetics/pathogenicity
Animals
Virulence/genetics
Polymerase Chain Reaction/methods
Macrophages/microbiology
Humans

@article {pmid39992407,
year = {2025},
author = {Zhang, W and Li, S and Xu, W and Wang, Q and Zhang, H and Liu, X and Chen, X and Xu, D and Chen, H},
title = {Knocking out artificially selected gene GmAOC4[H8] improves germination in soybean.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {3},
pages = {54},
pmid = {39992407},
issn = {1432-2242},
support = {32201845//National Natural Science Foundation of China/ ; 32001455//National Natural Science Foundation of China/ ; ZSBBL-KY2023-03//the Funding of Zhongshan Biological Breeding Laboratory/ ; BK20210154//Natural Science Foundation of Jiangsu Province/ ; JBGS[2021]060//the Open Competition Project of Seed Industry Revitalization of Jiangsu Province/ ; },
mesh = {*Glycine max/genetics/growth & development/physiology ; *Germination/genetics ; *Gene Expression Regulation, Plant ; *Oxylipins/metabolism ; *Cyclopentanes/metabolism ; *Seeds/growth & development/genetics ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Genes, Plant ; },
abstract = {Seed germination is an essential stage in the life cycle of flowering plants, influencing the field emergence rates of seeds. Consequently, the role of GmAOC4 in soybean seed germination was investigated in the present study. Results suggested that the chloroplast-localized GmAOC4 exhibited high expression levels in the roots and young pods and during the seed germination stage in soybeans. It was found that GmAOC4 has been artificially selected during soybean domestication and improvement and that GmAOC4[H8] showed repressed seed germination, of which the frequency in landraces and cultivars decreased when compared with wild soybean. Knocking out GmAOC4[H8] via CRISPR/Cas9 led to enhanced germination in gmaoc4 mutants, suggesting its negative regulation on seed germination in soybeans. Additionally, decreased endogenous jasmonic acid (JA) and JA precursor, 12-oxo-phytodienoic acid, were found in gmaoc4 mutants. RNA-seq analyses revealed that 91 and 269 differentially expressed genes (DEGs) were up-regulated and down-regulated in gmaoc4 mutants, respectively. Among these DEGs, three genes were involved in JA biosynthetic and signaling pathways. Our results offer new insights into the mechanism of soybean seed germination regulation by GmAOC4.},
}

*Glycine max/genetics/growth & development/physiology
*Germination/genetics
*Gene Expression Regulation, Plant
*Oxylipins/metabolism
*Cyclopentanes/metabolism
*Seeds/growth & development/genetics
Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Genes, Plant

@article {pmid39965366,
year = {2025},
author = {Cao, Y and Yang, Q and Guo, Y and Wang, X and Li, X and Zhang, N and Lu, W and Li, J and Zhang, X and Cao, L and Gong, P},
title = {An RPA-CRISPR/Cas12a-assisted method for nucleic acid detection of Haemonchus contortus in sheep.},
journal = {Veterinary parasitology},
volume = {334},
number = {},
pages = {110421},
doi = {10.1016/j.vetpar.2025.110421},
pmid = {39965366},
issn = {1873-2550},
mesh = {Animals ; *Haemonchus/genetics ; Sheep ; *Haemonchiasis/veterinary/diagnosis/parasitology ; *Sheep Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/veterinary ; *Sensitivity and Specificity ; Feces/parasitology ; Recombinases/metabolism/genetics ; },
abstract = {Haemonchus contortus (H. contortus), a highly pathogenic and blood-feeding nematode, could cause haemonchosis，resulting in tens of billions of dollars in production losses and significantly impacting the development of sheep husbandry. Rapid and accurate detection methods were particularly important for the prevention and control of haemonchosis. In this study, we developed a one-pot effective detection method that integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology based on the conserved region of ITS2 of H. contortus, with readout through fluorescence signals visualized by lateral flow strips (LFS) and observable under UV or blue light. The detection procedure was successfully finished in within 1 h and demonstrated high specificity and sensitivity, with no cross-reactivity detected with nine other common ovine pathogens and a detection limit as low as 0.1 copies/μL for fluorescence and 100 copies/μL for LFS. Validation with 89 sheep fecal samples revealed a 46.07 % positivity rate, fully consistent with quantitative PCR results. In summary, the RPA-CRISPR/Cas12a method for H. contortus detection exhibited the advantages of high specificity, high sensitivity, and low device dependence, portable and visible results. The technique presented significant potential for large-scale clinical application and provided novel point-of-care testing for clinical use in remote rural and resource-constrained areas.},
}

Animals
*Haemonchus/genetics
Sheep
*Haemonchiasis/veterinary/diagnosis/parasitology
*Sheep Diseases/diagnosis/parasitology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/veterinary
*Sensitivity and Specificity
Feces/parasitology
Recombinases/metabolism/genetics

@article {pmid39946489,
year = {2025},
author = {Du, Q and Zhang, H and Bi, Y and Wang, H and Zhou, X and Shi, P and Lv, S and Bi, S},
title = {N-Deficient B-Doped g-C3N4/CdS Heterojunction-Based PEC-FL Biosensor Assisted by CRISPR-Cas12a System for Ultrasensitive Determination of microRNA.},
journal = {Analytical chemistry},
volume = {97},
number = {7},
pages = {4049-4056},
doi = {10.1021/acs.analchem.4c05841},
pmid = {39946489},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Cadmium Compounds/chemistry ; *Sulfides/chemistry ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; Infrared Rays ; Bacterial Proteins ; Nitrogen Compounds ; CRISPR-Associated Proteins ; Graphite ; Endodeoxyribonucleases ; },
abstract = {Near-infrared light (NIR)-driven photoelectrochemical (PEC) processes are mainly faced with the limitation of weak photocurrents. Here, N-deficient B-doped g-C3N4/CdS (NB-g-C3N4/CdS) is proposed to construct a NIR-driven PEC biosensor assisted by CRISPR-Cas12a system for the determination of microRNA-21 (miRNA-21). To promote the optical absorption as well as the separation of photogenerated electrons and holes of g-C3N4, NB-g-C3N4/CdS is constructed via engineering the electronic and band structure in terms of N defect, B doping, and heterojunction, achieving high PEC performance. To obtain the high luminescence efficiency for exciting NB-g-C3N4/CdS under NIR, the core-shell NaYF4:Yb[3+], Tm[3+]@NaYF4 upconversion nanoparticles (UCNPs) with repaired defects are prepared. Furthermore, the rolling circle amplification (RCA)-assisted CRISPR-Cas12a system is integrated to fragment the DNA on UCNPs, achieving sensitive detection of miRNA-21. On the one hand, the uncleavaged signal probes on UCNPs combined with NB-g-C3N4/CdS through π-π stacking interaction, generating photocurrents under the irradiation of NIR. On the other hand, the cleavaged signal probes which cannot link with NB-g-C3N4/CdS exhibited the fluorescence (FL) signals. The proposed PEC-FL dual-mode biosensor provides a mutual authentication of testing results and demonstrates ultrasensitivity (the detection limit of 1.1 fM for PEC mode and 7.0 fM for FL mode) and excellent specificity, which is promising in the clinical analysis of miRNA.},
}

*MicroRNAs/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Cadmium Compounds/chemistry
*Sulfides/chemistry
Electrochemical Techniques/methods
Humans
Limit of Detection
Infrared Rays
Bacterial Proteins
Nitrogen Compounds
CRISPR-Associated Proteins
Graphite
Endodeoxyribonucleases

@article {pmid39937146,
year = {2025},
author = {Li, QN and Cui, YX and Dai, ZQ and Yao, ZL and Li, MY and Cai, QL and Kong, DM},
title = {Activator Strand Modifications in CRISPR/Cas12a: Unlocking the Potential for Casp-3-Targeted Biosensing and Imaging Analysis of Apoptosis.},
journal = {Analytical chemistry},
volume = {97},
number = {7},
pages = {4194-4201},
doi = {10.1021/acs.analchem.4c06591},
pmid = {39937146},
issn = {1520-6882},
mesh = {*Caspase 3/metabolism ; *Apoptosis/drug effects ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA/chemistry ; },
abstract = {The CRISPR/Cas12a system has emerged as a powerful tool in biosensing due to its unique trans-cleavage activity. This study conducted an in-depth investigation of the modulatory capabilities of this system, particularly focusing on the 5'-end modifications of the activator strand, and found that introducing a hairpin structure (HP) at the 5'-end of the activator strand, which was designed based on the RESET effect, can effectively suppress the activator strand's ability to activate the trans-cleavage activity of the CRISPR/Cas12a system. This suppression is independent of the HP's relation to the activator strand and the type of linker used (DNA, RNA or peptide). Detaching the HP from the activator strand restores the system's activity. These findings enrich the development of CRISPR/Cas12a-based biosensors, and expand their application beyond DNA-based target detection to peptide sequence-based target recognition. Based on this discovery, we constructed a sensitive biosensor for caspase-3 (Casp-3), a key executor in apoptosis, by linking the HP to the activator strand with a peptide linker containing a Casp-3 recognition site. The proposed biosensor has been validated for its sensitivity and specificity in detecting Casp-3, as well as for monitoring drug-induced apoptosis through the imaging of Casp-3 in living cells, providing a valuable tool for studying the apoptotic process, screening drugs, assessing drug efficacy, and evaluating treatment outcomes. This strategy also shows promise for detecting other peptide-based targets, broadening the horizons for early disease biomarker detection and timely therapeutic interventions.},
}

*Caspase 3/metabolism
*Apoptosis/drug effects
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
DNA/chemistry

@article {pmid39919527,
year = {2025},
author = {Zhou, Y and Chen, Y and Song, X and Zhong, Z and Guo, Q and Jing, S and Ayanniyi, OO and Lu, Z and Zhang, Q and Yang, C},
title = {Rapid and sensitive detection of Trichomonas gallinae using RAA-CRISPR-Cas12a.},
journal = {Veterinary parasitology},
volume = {334},
number = {},
pages = {110412},
doi = {10.1016/j.vetpar.2025.110412},
pmid = {39919527},
issn = {1873-2550},
mesh = {*Trichomonas/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; *Trichomonas Infections/veterinary/diagnosis/parasitology ; *Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods/veterinary ; Poultry Diseases/parasitology/diagnosis ; CRISPR-Associated Proteins/genetics ; Recombinases/metabolism/genetics ; },
abstract = {Trichomonas gallinae (T. gallinae) is an important pathogen causing trichomoniasis in birds, especially pigeons. Rapid and sensitive detection methods for T. gallinae are urgently needed to diagnose T. gallinae early to reduce poultry industry losses. Therefore, we developed a rapid and sensitive diagnostic method based on recombinase-aided amplification (RAA) assay and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (CRISPR/Cas12a) system to detect T. gallinae. The RAA-CRISPR/Cas12a method can be divided into RAA-CRISPR/Cas12a fluorescent signal (RAA-CRISPR/Cas12a-FL) and RAA-CRISPR/Cas12a lateral flow strip (RAA-CRISPR/Cas12-LFS). Both RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12-LFS methods show the property of rapid. sensitive, and does not require a sophisticated instrument, and they allow the detection of T. gallinae in less than 1 hr. Meanwhile, they have satisfactory specificity and can accurately detect T. gallinae in samples of different pathogens. In summary, the RAA-CRISPR/Cas12a-FL and RAA-CRISPR/Cas12-LFS methods we constructed can be used for on-site T. gallinae detection and resource-poor areas.},
}

*Trichomonas/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
*Trichomonas Infections/veterinary/diagnosis/parasitology
*Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods/veterinary
Poultry Diseases/parasitology/diagnosis
CRISPR-Associated Proteins/genetics
Recombinases/metabolism/genetics

@article {pmid39919116,
year = {2025},
author = {Juan, T and Molina, T and Xie, L and Papadopoulou, S and Cardoso, B and Jha, SG and Stainier, DYR},
title = {A recombinase-activated ribozyme to knock down endogenous gene expression in zebrafish.},
journal = {PLoS genetics},
volume = {21},
number = {2},
pages = {e1011594},
doi = {10.1371/journal.pgen.1011594},
pmid = {39919116},
issn = {1553-7404},
mesh = {*Zebrafish/genetics ; Animals ; *RNA, Catalytic/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Knockdown Techniques ; RNA, Messenger/genetics/metabolism ; Gene Editing/methods ; Introns/genetics ; Zebrafish Proteins/genetics/metabolism ; Recombinases/genetics/metabolism ; DNA Nucleotidyltransferases/genetics/metabolism ; },
abstract = {Precise regulation of gene expression is essential to understand a wide range of biological processes. Control over gene expression can be achieved using site-directed recombinases and endonucleases whose efficiency is variable and dependent on the genomic context. Here, we develop a self-cleaving ribozyme-based tool to control mRNA levels of endogenous targets in zebrafish. Using an in vivo reporter strategy, we first show that inserting the T3H48 self-cleaving ribozyme in an intron enables rapid pre-mRNA cleavage, with up to 20-fold reduction in expression, and that this ribozyme displays superior activity compared with other ribozymes. We then inserted the T3H48 ribozyme in the second intron of the albino gene using a CRISPR/Cas9 strategy and observed a pigmentation phenotype similar to that in the mutant. Using a base-editing strategy to inactivate the ribozyme, we also show that this phenotype is reversible, illustrating the specificity of the approach. In addition, we generated a Flippase- and Cre-activatable version of the T3H48 ribozyme, called RiboFlip, to control the mRNA levels of the albino gene. RiboFlip activation induced mRNA knockdown and also recapitulated the albino mutant phenotype. Furthermore, we show that a Cre- and Dre-controllable Gal4/UAS reporter in the RiboFlip cassette can label knocked-down cells independently of the expression of the target gene. Altogether, we introduce the RiboFlip cassette as a flexible tool to control endogenous gene expression in a vertebrate model and as an alternative to existing conditional knockdown strategies.},
}

*Zebrafish/genetics
Animals
*RNA, Catalytic/genetics/metabolism
*CRISPR-Cas Systems
Gene Knockdown Techniques
RNA, Messenger/genetics/metabolism
Gene Editing/methods
Introns/genetics
Zebrafish Proteins/genetics/metabolism
Recombinases/genetics/metabolism
DNA Nucleotidyltransferases/genetics/metabolism

@article {pmid39902531,
year = {2025},
author = {Lafi, Z and Ata, T and Asha, S},
title = {CRISPR in clinical diagnostics: bridging the gap between research and practice.},
journal = {Bioanalysis},
volume = {17},
number = {4},
pages = {281-290},
doi = {10.1080/17576180.2025.2459520},
pmid = {39902531},
issn = {1757-6199},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Polymorphism, Single Nucleotide ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) has transformed molecular biology through its precise gene-editing capabilities. Beyond its initial applications in genetic modification, CRISPR has emerged as a powerful tool in diagnostics and biosensing. This review explores its transition from genome editing to innovative detection methods, including nucleic acid identification, single nucleotide polymorphism (SNP) analysis, and protein sensing. Advanced technologies such as SHERLOCK and DETECTR demonstrate CRISPR's potential for point-of-care diagnostics, enabling rapid and highly sensitive detection. The integration of chemical modifications, CRISPR-Chip technology, and enzymatic systems like Cas12a and Cas13a enhances signal amplification and detection efficiency. These advancements promise decentralized, real-time diagnostic solutions with significant implications for global healthcare. Furthermore, the fusion of CRISPR with artificial intelligence and digital health platforms is paving the way for more accessible, cost-effective, and scalable diagnostic approaches, ultimately revolutionizing precision medicine.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Polymorphism, Single Nucleotide

@article {pmid39899469,
year = {2025},
author = {Dong, J and Croslow, SW and Lane, ST and Castro, DC and Blanford, J and Zhou, S and Park, K and Burgess, S and Root, M and Cahoon, EB and Shanklin, J and Sweedler, JV and Zhao, H and Hudson, ME},
title = {Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping.},
journal = {The Plant cell},
volume = {37},
number = {2},
pages = {},
doi = {10.1093/plcell/koaf026},
pmid = {39899469},
issn = {1532-298X},
support = {//Center for Advanced Bioenergy/ ; //U.S. Department of Energy/ ; },
mesh = {*Zea mays/genetics/metabolism ; *Gene Editing/methods ; *Phenotype ; *Nicotiana/genetics/metabolism/cytology ; *Lipids ; Plant Cells/metabolism ; Genome, Plant ; Protoplasts/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods ; Genetic Engineering/methods ; },
abstract = {Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.},
}

*Zea mays/genetics/metabolism
*Gene Editing/methods
*Phenotype
*Nicotiana/genetics/metabolism/cytology
*Lipids
Plant Cells/metabolism
Genome, Plant
Protoplasts/metabolism
CRISPR-Cas Systems
Plants, Genetically Modified
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods
Genetic Engineering/methods

@article {pmid39842606,
year = {2025},
author = {Zhang, K and Wang, Y and Jiang, S and Li, Y and Xiang, P and Zhang, Y and Chen, Y and Chen, M and Su, W and Liu, L and Li, S},
title = {dsDAP: An efficient method for high-abundance DNA-encoded library construction in mammalian cells.},
journal = {International journal of biological macromolecules},
volume = {298},
number = {},
pages = {140089},
doi = {10.1016/j.ijbiomac.2025.140089},
pmid = {39842606},
issn = {1879-0003},
mesh = {Humans ; HEK293 Cells ; *Gene Library ; *DNA/genetics ; *CRISPR-Cas Systems ; Polymerase Chain Reaction/methods ; },
abstract = {DNA-encoded libraries are invaluable tools for high-throughput screening and functional genomics studies. However, constructing high-abundance libraries in mammalian cells remains challenging. Here, we present dsDNA-assembly-PCR (dsDAP), a novel Gibson-assembly-PCR strategy for creating DNA-encoded libraries, offering improved flexibility and efficiency over previous methods. We demonstrated this approach by investigating the impact of translation initiation sequences (TIS) on protein expression in HEK293T cells. Both CRISPR-Cas9 and piggyBac systems were employed for genomic integration, allowing comparison of different integration methods. Our results confirmed the importance of specific nucleotides in the TIS region, particularly the preference for adenine at the -3 position in high-expression sequences. We also explored the effects of library dilution on genotype-phenotype correlations. This Gibson-assembly-PCR strategy overcomes limitations of existing methods, such as restriction enzyme dependencies, and provides a versatile tool for constructing high-abundance libraries in mammalian cells. Our approach has broad applications in functional genomics, drug discovery, and the study of gene regulation.},
}

Humans
HEK293 Cells
*Gene Library
*DNA/genetics
*CRISPR-Cas Systems
Polymerase Chain Reaction/methods

@article {pmid39838754,
year = {2025},
author = {Zhang, D and Zhou, Y and Li, X and Luan, Q},
title = {CRISPR/Cas13a-Enhanced Porous Hydrogel Encapsulated Photonic Barcodes for Multiplexed Detection of Virus.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {8},
pages = {e2408725},
doi = {10.1002/smll.202408725},
pmid = {39838754},
issn = {1613-6829},
support = {82102511//National Natural Science Foundation of China/ ; 82102181//National Natural Science Foundation of China/ ; BK20210021//National Natural Science Foundation of China/ ; BK20210009//National Natural Science Foundation of China/ ; M2021031//Research Project of Jiangsu Province Health Committee/ ; 2024-LCYJ-MS-15//Medical School of Nanjing University/ ; (YKK23068)//Nanjing Medical Science and Technique Development Foundation YKK23068/ ; },
mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/genetics/isolation & purification ; *Hydrogels/chemistry ; Porosity ; Humans ; Photons ; COVID-19/diagnosis/virology ; RNA, Viral/genetics ; Nucleic Acid Hybridization ; },
abstract = {In this study, we present an ultrasensitive and specific multiplexed detection method for SARS-CoV-2 and influenza (Flu) utilizing CRISPR/Cas13a technology combined with a hydrogel-encapsulated photonic crystal (PhC) barcode integrated with hybridization chain reaction (HCR). The barcodes, characterized by core-shell structures, are fabricated through partial replication of periodically ordered hexagonally close-packed silicon dioxide beads. Consequently, the opal hydrogel shell of these barcodes features abundant interconnected pores that provide a substantial surface area for probe immobilization. Furthermore, the inherent structural colors remain stable during detection events due to the robust mechanical strength of the barcode cores. This integration of CRISPR/Cas13a and HCR leverages both the highly specific RNA recognition capabilities and trans-cleavage activity of Cas13a while employing HCR to enhance sensitivity. Upon encountering target RNA, Cas13a cleaves a hairpin probe, thereby initiating subsequent HCR amplification for enhanced detection sensitivity. Our method demonstrates high accuracy and sensitivity in multiplexed detection of SARS-CoV-2, Flu A and Flu B RNA with a limit-of-detection as low as 200 aM. Importantly, this assay also exhibits acceptable accuracy in repeated clinical sample testing. Thus, our platform represents a promising strategy for highly sensitive multiplexed virus detection in clinical.},
}

*CRISPR-Cas Systems
*SARS-CoV-2/genetics/isolation & purification
*Hydrogels/chemistry
Porosity
Humans
Photons
COVID-19/diagnosis/virology
RNA, Viral/genetics
Nucleic Acid Hybridization

@article {pmid39824412,
year = {2025},
author = {Li, L and Li, J and Wang, S and Dong, Y},
title = {Dual-mode CRISPR/Cas12a-assisted fluorescent and lateral flow aptasensor based on a newly truncated aptamer for Fumonisin B1 detection.},
journal = {International journal of biological macromolecules},
volume = {298},
number = {},
pages = {139950},
doi = {10.1016/j.ijbiomac.2025.139950},
pmid = {39824412},
issn = {1879-0003},
mesh = {*Fumonisins/analysis ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; CRISPR-Associated Proteins/genetics/chemistry ; Bacterial Proteins/genetics/chemistry ; Endodeoxyribonucleases/chemistry/genetics/metabolism ; },
abstract = {As a Group 2B carcinogen, accurate and efficient detection for Fumonisin B1 (FB1) is essential. The emergence of aptamers presents a viable solution to meet this demand. In this study, a truncated aptamer named Apt40 was developed, showcasing remarkable binding affinity to FB1. In recent years, the role of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated 12a protein (Cas12a) in detection became increasingly significant, especially utilizing the trans-cleavage of Cas12a/CRISPR RNA (crRNA) complex. To further evaluate the applicability of the Apt40, a dual-mode CRISPR/Cas12a-assisted fluorescent and lateral flow aptasensor was constructed. Notably, the crRNA was designed to complementarily bind with Apt40 in its active binding sites, thus activating the trans-cleavage of Cas12a/crRNA to signal probes. Consequently, the fluorescent aptasensor exhibited a linear range of 10-1500 ng/mL with a Limit of Detection (LOD) of 0.802 ng/mL, while the lateral flow aptasensor showed a 200-3000 ng/mL linear range with a 9.031 ng/mL LOD. Both aptasensors provided high recoveries (95.11 %-106.63 %) in corn oil and starch samples, underscoring their precisions. We anticipate that this systematic strategy, from optimizing aptamer's performance to developing a novel dual-mode aptasensor, can provide a universal framework and valuable insights for the detection of other target molecules.},
}

*Fumonisins/analysis
*Aptamers, Nucleotide/chemistry
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
CRISPR-Associated Proteins/genetics/chemistry
Bacterial Proteins/genetics/chemistry
Endodeoxyribonucleases/chemistry/genetics/metabolism

@article {pmid39800033,
year = {2025},
author = {Guo, Q and Huang, L and Liu, Y and Qi, C and Shao, H and Peng, J and Zhang, H and Zang, Y and Luo, M and Shen, X and Wang, D and Liu, L and Xu, Q and Zheng, Y and Dong, X and Xu, X},
title = {PcoCas12a: A novel CRISPR enzyme from Prevotella copri enhancing TCR-T-cell tumor suppression.},
journal = {International journal of biological macromolecules},
volume = {298},
number = {},
pages = {139740},
doi = {10.1016/j.ijbiomac.2025.139740},
pmid = {39800033},
issn = {1879-0003},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *T-Lymphocytes/immunology ; *Prevotella/genetics ; *Receptors, Antigen, T-Cell/genetics/immunology ; Animals ; Mice ; Gene Knockout Techniques ; Cell Line, Tumor ; },
abstract = {Genome editing technologies have been widely utilized in cell engineering, demonstrating immense potential in cell and gene therapy. However, an optimal gene-editing enzyme for immune cell editing remains unidentified. In this study, we identified a novel gene editing enzyme, termed CRISPR/PcoCas12a, derived from Prevotella copri, which recognizes a 5'-YYN PAM sequence. We demonstrated that CRISPR/PcoCas12a offers a broader range of editing sites and superior editing efficiency at specific loci compared to AsCas12a. Furthermore, we illustrated its capability to enhance tumor suppression by targeting DGKα in TCR-T cells. DGKα functions as a negative regulator of T cell function, and its knockout significantly boosts the antitumor efficacy of TCR-T cells. The knockout efficiency and tumor suppressor ability of PcoCas12a targeting DGKα were markedly higher than those achieved with AsCas12a. Single-cell sequencing data confirmed that PcoCas12a-mediated DGKα gene knockout improves the tumor suppressive capabilities of T cells by promoting T-cell activation and strengthening immune regulatory responses. These findings establish PcoCas12a as a highly efficient enzyme for T cell editing, indicating its potential application in T-cell therapy.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*T-Lymphocytes/immunology
*Prevotella/genetics
*Receptors, Antigen, T-Cell/genetics/immunology
Animals
Mice
Gene Knockout Techniques
Cell Line, Tumor

@article {pmid39696792,
year = {2024},
author = {Zhao, Y and Li, Z and Li, T and Rao, R and Zhu, J and Hu, R and Xu, G and Li, Y and Yang, Y},
title = {SlipChip Enables the Integration of CRISPR-Cas12a and RPA for Fast and Stand-Alone HPV Detection.},
journal = {Analytical chemistry},
volume = {96},
number = {52},
pages = {20602-20611},
doi = {10.1021/acs.analchem.4c05290},
pmid = {39696792},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 16/genetics ; DNA, Viral/analysis/genetics ; Human papillomavirus 18/genetics ; Papillomavirus Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Female ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Human papillomavirus (HPV) screening is vital for the early detection and prevention of cervical cancer. However, existing methods often face challenges related to speed, simplicity, and multiplexing, especially in resource-limited settings. Here we developed a portable SlipChip-based multiplexed and rapid nucleic acid testing platform, named SMART, designed to simultaneously detect HPV16 and HPV18. SMART allows seamless integration of the RPA and Cas12a assays on the SlipChip and includes a heating membrane to regulate the on-chip assay temperatures. This allows SMART to operate as a stand-alone platform without additional control instruments. The platform also features an All-in-One imaging mode for rapid on-chip data acquisition, enhancing its performance. SMART enables sensitive detection of HPV16 and HPV18 DNA across multiple samples in just 36 min with a detection limit of approximately 6 copies per reaction. Testing of 56 clinical samples at risk of HPV infection validated SMART's performance, showing 97.7% sensitivity and 100% specificity. In summary, SMART offers a stand-alone system capable of rapidly distinguishing between the two most harmful HPV subtypes, showcasing the significant potential for rapid, multiplexed nucleic acid testing in various applications.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human papillomavirus 16/genetics
DNA, Viral/analysis/genetics
Human papillomavirus 18/genetics
Papillomavirus Infections/diagnosis/virology
Nucleic Acid Amplification Techniques/methods
Female
CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39991929,
year = {2025},
author = {Herrmann May, N and Cao, A and Schmid, A and Link, F and Arias-Del-Angel, J and Meiser, E and Beneke, T},
title = {Improved base editing and functional screening in Leishmania via co-expression of the AsCas12a ultra variant, a T7 RNA polymerase, and a cytosine base editor.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
doi = {10.7554/eLife.97437},
pmid = {39991929},
issn = {2050-084X},
support = {ALTF 727-2021//European Molecular Biology Organization/ ; 101064428 - LeishMOM//HORIZON EUROPE Marie Sklodowska-Curie Actions/ ; 532631727//Deutsche Forschungsgemeinschaft/ ; Project D3 and B3//LOEWE Center DRUID/ ; },
mesh = {*Gene Editing/methods ; *Leishmania/genetics/metabolism ; *DNA-Directed RNA Polymerases/metabolism/genetics ; *Cytosine/metabolism ; Viral Proteins/genetics/metabolism ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; },
abstract = {The ability to analyze the function of all genes in a genome is highly desirable, yet challenging in Leishmania due to a repetitive genome, limited DNA repair mechanisms, and lack of RNA interference in most species. While our introduction of a cytosine base editor (CBE) demonstrated potential to overcome these limitations (Engstler and Beneke, 2023), challenges remained, including low transfection efficiency, variable editing rates across species, parasite growth effects, and competition between deleterious and non-deleterious mutations. Here, we present an optimized approach addressing these issues. We identified a T7 RNAP promoter variant ensuring high editing rates across Leishmania species without compromising growth. A revised CBE single-guide RNAs (sgRNAs) scoring system was developed to prioritize STOP codon generation. Additionally, a triple-expression construct was created for stable integration of CBE sgRNA expression cassettes into a Leishmania safe harbor locus using AsCas12a ultra-mediated DNA double-strand breaks, increasing transfection efficiency by ~400-fold to 1 transfectant per 70 transfected cells. Using this improved system for a small-scale proof-of-principle pooled screen, we successfully confirmed the essential and fitness-associated functions of CK1.2, CRK2, CRK3, AUK1/AIRK, TOR1, IFT88, IFT139, IFT140, and RAB5A in Leishmania mexicana, demonstrating a significant improvement over our previous method. Lastly, we show the utility of co-expressing AsCas12a ultra, T7 RNAP, and CBE for hybrid CRISPR gene replacement and base editing within the same cell line. Overall, these improvements will broaden the range of possible gene editing applications in Leishmania species and will enable a variety of loss-of-function screens in the near future.},
}

*Gene Editing/methods
*Leishmania/genetics/metabolism
*DNA-Directed RNA Polymerases/metabolism/genetics
*Cytosine/metabolism
Viral Proteins/genetics/metabolism
CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism

@article {pmid39990123,
year = {2025},
author = {Lee, M and Kim, Y and Lee, HW and Park, Y and Yi, S},
title = {Complete genome sequence of psychrobacter sp. KFRI-CH2-11: A psychrotolerant bacterium with probiotic, biofortification, and antimicrobial potential for the dairy and meat industries.},
journal = {Data in brief},
volume = {59},
number = {},
pages = {111344},
pmid = {39990123},
issn = {2352-3409},
abstract = {This dataset provides the complete genome sequence of Psychrobacter sp. KFRI-CH2-11, isolated from Korean fermented anchovy, Myeolchi-jeotgal. Genomic analysis identified genes involved in Vitamin B12 biosynthesis, carbohydrate metabolism, CRISPR-Cas defense systems, and antioxidant activity, underscoring the strain's potential for use in food biotechnology. Additional genes linked to antibiotic resistance and bioremediation suggest adaptability in diverse environments, particularly cold-chain storage in the dairy and meat industry. PathogenFinder analysis confirmed the absence of pathogenicity-associated genes, validating the strain's suitability as a probiotic and biofortifying agent in food products.},
}