@article {pmid40424009,
year = {2025},
author = {Wei, L and Wang, Z and She, Y and Fu, H},
title = {CRISPR/Cas Multiplexed Biosensing: Advances, Challenges, and Perspectives.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c04428},
pmid = {40424009},
issn = {1520-6882},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems are renowned for their high sensitivity and specificity, enabling them as a powerful diagnostic toolbox. Multiplexed detection of panels of targets, as opposed to single targets, is imperative for reliable and conclusive disease diagnostics. However, multiplex application of the CRISPR/Cas system has long been hindered by indistinguishable signals from specific targets due to nonspecific chaotic trans-cleavage. To make a breakthrough, substantial efforts have been devoted to CRISPR/Cas-powered multiplexed biosensing strategies, which consequently experienced rapid development over the past five years. This review systematically summarizes recent advances in CRISPR/Cas multiplexed detection encompassing Cas9, Cas12, and Cas13. Key focus issues include multiplex biosensing strategies and their respective advantages and limitations, sensing mechanisms, and detection performance of novel validated examples. Finally, the status and challenges of CRISPR/Cas multiplexed biosensing are critically discussed, and future outlooks are proposed for their potential practical application. This Perspective aims to inspire significant research and promote the development of the next generation of CRISPR/Cas multiplexed biosensing.},
}

@article {pmid40422684,
year = {2025},
author = {Ma, B and Li, Y and Wang, T and Li, D and Jia, S},
title = {Advances in CRISPR/Cas9-Based Gene Editing in Filamentous Fungi.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {11},
number = {5},
pages = {},
pmid = {40422684},
issn = {2309-608X},
support = {32160143//National Science Foundation of China/ ; },
abstract = {As an important class of microorganisms, filamentous fungi have crucial roles in protein secretion, secondary metabolite production and environmental pollution control. However, characteristics such as apical growth, heterokaryon, low homologous recombination (HR) efficiency and the scarcity of genetic markers mean that the application of traditional gene editing technology in filamentous fungi faces great challenges. The introduction of the RNA-mediated CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRlSPR-associated protein) system in filamentous fungi in recent years has revolutionized gene editing in filamentous fungi. In addition, the continuously expressed CRISPR system has significantly improved the editing efficiency, while the optimized sgRNA design and reduced cas9 concentration have effectively reduced the off-target effect, further enhancing the safety and reliability of the technology. In this review, we systematically analyze the molecular mechanism and regulatory factors of CRISPR/Cas9, focus on the optimization of its expression system and the improvement of the transformation efficiency in filamentous fungi, and reveal the core regulatory roles of HR and non-homologous end-joining (NHEJ) pathways in gene editing. Based on the analysis of various filamentous fungi applications, this review reveals the outstanding advantages of CRISPR/Cas9 in the enhancement of protein secretion, addresses the reconstruction of secondary metabolic pathways and pollutant degradation in the past decade, and provides a theoretical basis and practical guidance for the optimization of the technology and engineering applications.},
}

@article {pmid40422214,
year = {2025},
author = {Gibson, J and Dhungana, A and Pokhrel, M and Arthur, B and Suresh, P and Adebayo, O and Cottle, RN},
title = {Validation of Clinical-Grade Electroporation Systems for CRISPR-Cas9-Mediated Gene Therapy in Primary Hepatocytes for the Correction of Inherited Metabolic Liver Disease.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422214},
issn = {2073-4409},
support = {1R01HL168093-01A1/GF/NIH HHS/United States ; 2021000920//American Association for the Study of Liver Diseases Foundation/ ; R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism ; *Electroporation/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Mice ; Gene Editing/methods ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver Diseases/therapy/genetics ; Disease Models, Animal ; Male ; Hydrolases ; },
abstract = {Hepatocyte transplantation (HTx) combined with ex vivo gene therapy has garnered significant interest due to its potential for treating many inherited metabolic liver diseases. The biggest obstacle for HTx is achieving sufficient engraftment levels to rescue diseased phenotypes, which becomes more challenging when combined with ex vivo gene editing techniques. However, recent technological advancements have improved electroporation delivery efficiency, cell viability, and scalability for cell therapy. We recently demonstrated the impacts of electroporation for cell-based gene therapy in a mouse model of hereditary tyrosinemia type 1 (HT1). Here, we explore the use of the clinical-grade electroporator, the MaxCyte ExPERT GTx, utilized in the first FDA-approved CRISPR therapy, Casgevy, and evaluate its potential in primary hepatocytes in terms of delivery efficiency and cell viability. We assessed the gene editing efficiency and post-transplantation engraftment of hepatocytes from mTmG mice electroporated with CRISPR-Cas9-ribonucleoproteins (RNPs) targeting 4-hydroxyphenylpyruvate dioxygenase (Hpd) in a fumarylacetoacetate hydrolase (Fah)-deficient mouse model of HT1. After surgery, Fah[-/-] graft recipients were cycled off and on nitisinone to achieve independence from drug-induced Hpd inhibition, an indicator of HT1 disease correction. Transplanted hepatocytes subjected to electroporation using the GTx system had a cell viability of 89.9% and 100% on-target gene editing efficiency. Recipients transplanted with GTx-electroporated cells showed a smaller weight reduction than controls transplanted with untransfected cells (7.9% and 13.8%, respectively). Further, there were no mortalities in the GTx-recipient mice, whereas there was 25% mortality in the control recipients. Mean donor cell engraftment was significantly higher in GTx-recipient mice compared to untransfected control recipients (97.9% and 81.6%, respectively). Our results indicate that the GTx system does not negatively impact hepatocyte functionality and engraftment potential, thereby demonstrating the promise of GTx electroporation in hepatocytes as a viable cell therapy for treating genetic diseases that affect the liver.},
}

*Hepatocytes/transplantation/metabolism
*Electroporation/methods
Animals
*CRISPR-Cas Systems/genetics
*Genetic Therapy/methods
Mice
Gene Editing/methods
*Tyrosinemias/therapy/genetics
Humans
*Liver Diseases/therapy/genetics
Disease Models, Animal
Male
Hydrolases

@article {pmid40422195,
year = {2025},
author = {Orlova, NA and Sinegubova, MV and Kolesov, DE and Khodak, YA and Tatarskiy, VV and Vorobiev, II},
title = {Genomic and Phenotypic Characterization of CHO 4BGD Cells with Quad Knockout and Overexpression of Two Housekeeping Genes That Allow for Metabolic Selection and Extended Fed-Batch Culturing.},
journal = {Cells},
volume = {14},
number = {10},
pages = {},
pmid = {40422195},
issn = {2073-4409},
support = {25-24-00140//Russian Science Foundation/ ; },
mesh = {CHO Cells ; Animals ; Cricetulus ; *Batch Cell Culture Techniques/methods ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Gene Editing ; Apoptosis/genetics ; *Genes, Essential/genetics ; Phenotype ; *Genomics ; },
abstract = {Re-engineering of CHO cells using genome editing and the overexpression of multiple helper genes is the central track for obtaining better cell lines for the production of biopharmaceuticals. Using two subsequent rounds of genome editing of the CHO S cells, we have developed the cell line CHO 4BGD with four knockouts of two pro-apoptotic genes bak1 and bax, and two common selection markers genes-glul (GS) and dhfr, and additional copies of genes bcl-2 and beclin-1 used for enhancement of macroautophagy. The NGS sequencing of 4BGD cells revealed that all eight targeted alleles were successfully disrupted. Two edited loci out of eight contained large inserts of non-relevant DNA. Further data analysis shows that cells have no off-target DNA editing events, and all known CHO genes are preserved. The cells obtained are completely resistant to the induction of apoptosis, and they are suitable for the generation of stably transfected cell lines with the dhfr selection marker. They also properly undergo the target gene amplification. The 4BGD-derived clonal cell line that secretes the monoclonal antibody retains the ability for prolonged fed-batch culturing. The method of obtaining multiply edited CHO cells using the multiplex CRISPR/Cas9 editing and simultaneous stable transfection of plasmids, coding for the housekeeping genes, is suitable for the rapid generation of massively edited CHO cells.},
}

CHO Cells
Animals
Cricetulus
*Batch Cell Culture Techniques/methods
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
Gene Editing
Apoptosis/genetics
*Genes, Essential/genetics
Phenotype
*Genomics

@article {pmid40419487,
year = {2025},
author = {Byrne, SM and Burleigh, SM and Fragoza, R and Jiang, Y and Savva, Y and Pabon, R and Kania, E and Rainaldi, J and Portell, A and Mali, P and Briggs, AW},
title = {An engineered U7 small nuclear RNA scaffold greatly increases ADAR-mediated programmable RNA base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4860},
pmid = {40419487},
issn = {2041-1723},
mesh = {*Adenosine Deaminase/metabolism/genetics ; Animals ; *RNA Editing/genetics ; Humans ; Mice ; *RNA, Small Nuclear/genetics/metabolism/chemistry ; *Gene Editing/methods ; *RNA-Binding Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Custom RNA base editing exploiting the human Adenosine Deaminase Acting on RNA (ADAR) enzyme may enable therapeutic gene editing without DNA damage or use of foreign proteins. ADAR's adenosine-to-inosine (effectively A-to-G) deamination activity can be targeted to transcripts using an antisense guide RNA (gRNA), but efficacy is challenged by limits of in vivo delivery. Embedding gRNAs into a U7 small nuclear RNA (snRNA) framework greatly enhances RNA editing with endogenous ADAR, and a 750-plex single-cell mutagenesis screen further improved the framework. An optimized scaffold with a stronger synthetic U7 promoter enables 76% RNA editing in vitro from a single DNA construct per cell, and 75% editing in a Hurler syndrome mouse brain after one systemic AAV injection, surpassing circular gRNA approaches. The technology also improves published DMD exon-skipping designs 25-fold in differentiated myoblasts. Our engineered U7 framework represents a universal scaffold for ADAR-based RNA editing and other antisense RNA therapies.},
}

*Adenosine Deaminase/metabolism/genetics
Animals
*RNA Editing/genetics
Humans
Mice
*RNA, Small Nuclear/genetics/metabolism/chemistry
*Gene Editing/methods
*RNA-Binding Proteins/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid40419416,
year = {2025},
author = {Wachholz Junior, D and Pontes, RG and Hryniewicz, BM and Kubota, LT},
title = {Exploring a CRISPR/Cas12a-powered impedimetric biosensor for amplification-free detection of a pathogenic bacterial DNA.},
journal = {Biosensors & bioelectronics},
volume = {},
number = {},
pages = {117607},
doi = {10.1016/j.bios.2025.117607},
pmid = {40419416},
issn = {1873-4235},
abstract = {Timely and precise detection of bacterial infections is essential for improving patient outcomes and reducing healthcare costs, especially for sepsis, where delayed diagnosis increases mortality. Traditional culture- and PCR-based methods are time consuming and require complex sample processing, making them unsuitable for rapid diagnostics in resource-limited settings. CRISPR/Cas-based methods, particularly when combined with electrochemical sensing, offer a promising alternative for rapid point-of-care (POC) diagnostics of bacterial infections due to their simplicity and specificity. This study proposes a label-free impedimetric biosensor using the CRISPR/Cas12a system for rapid and amplification-free detection of Staphylococcus aureus DNA, a primary pathogen responsible for sepsis. By leveraging CRISPR/Cas12a's target-activated collateral cleavage on non-specific DNA reporters we investigated the impact of using a protospacer adjacent motif (PAM) sequence on detection sensitivity and specificity. Our biosensor demonstrated ultra-sensitive detection, with limit of detection as low as 20 aM for dsDNA targets in buffer and without any pre-amplification steps. The study also confirmed CRISPR specificity's dependence on the PAM sequence, showing that mismatches on targeting sequences reduces cleavage efficiency, with a drastic reduction in trans-cleavage activity for single mismatch in PAM-containing sequences. Additionally, we examined how the DNA reporter affects performance, noting reduced cleavage efficiency when a ssDNA target was paired with a dsDNA reporter. Furthermore, validation experiments using human serum samples confirmed the biosensor's accuracy for bacterial DNA detection in clinical settings. This work advances CRISPR-powered electrochemical biosensors, providing a detailed discussion on developing a highly sensitive, fast and amplification-free tool for early detection of sepsis-causing bacteria.},
}

@article {pmid40418657,
year = {2025},
author = {Johnson, MJ and DeFeo, AP and Slipek, NJ and Folsom, TD and Henley, T and Choudhry, MS and Webber, BR and Moriarity, BS},
title = {Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/68150},
pmid = {40418657},
issn = {1940-087X},
mesh = {Humans ; *T-Lymphocytes/cytology/immunology ; CRISPR-Cas Systems ; *DNA/genetics ; Animals ; *Cell Engineering/methods ; Mice ; *DNA End-Joining Repair ; *Genetic Engineering/methods ; },
abstract = {Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.},
}

Humans
*T-Lymphocytes/cytology/immunology
CRISPR-Cas Systems
*DNA/genetics
Animals
*Cell Engineering/methods
Mice
*DNA End-Joining Repair
*Genetic Engineering/methods

@article {pmid40417002,
year = {2025},
author = {Gallala, M},
title = {Application of CRISPR/Cas gene editing for infectious disease control in poultry.},
journal = {Open life sciences},
volume = {20},
number = {1},
pages = {20251095},
pmid = {40417002},
issn = {2391-5412},
abstract = {The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.},
}

@article {pmid40315964,
year = {2025},
author = {Gupta, MK and Gouda, G and Moazzam-Jazi, M and Vadde, R and Nagaraju, GP and El-Rayes, BF},
title = {CRISPR/Cas9-directed epigenetic editing in colorectal cancer.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1880},
number = {3},
pages = {189338},
doi = {10.1016/j.bbcan.2025.189338},
pmid = {40315964},
issn = {1879-2561},
mesh = {Humans ; *Colorectal Neoplasms/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Epigenesis, Genetic ; Animals ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Epigenome Editing ; },
abstract = {Colorectal cancer (CRC) remains a leading cause of cancer-related illness and death worldwide, arising from a complex interplay of genetic predisposition, environmental influences, and epigenetic dysregulation. Among these factors, epigenetic modifications-reversible and heritable changes in gene expression-serve as crucial regulators of CRC progression. Understanding these modifications is essential for identifying potential biomarkers for early diagnosis and developing targeted therapeutic strategies. Epigenetic drugs (epidrugs) such as DNA methyltransferase inhibitors (e.g., decitabine) and bromodomain inhibitors (e.g., JQ1) have shown promise in modulating aberrant epigenetic changes in CRC. However, challenges such as drug specificity, delivery, and safety concerns limit their clinical application. Advances in CRISPR-Cas9-based epigenetic editing offer a more precise approach to modifying specific epigenetic markers, presenting a potential breakthrough in CRC treatment. Despite its promise, CRISPR-based epigenome editing may result in unintended genetic modifications, necessitating stringent regulations and safety assessments. Beyond pharmacological interventions, lifestyle factors-including diet and gut microbiome composition-play a significant role in shaping the epigenetic landscape of CRC. Nutritional and microbiome-based interventions have shown potential in preventing CRC development by maintaining intestinal homeostasis and reducing tumor-promoting epigenetic changes. This review provides a comprehensive overview of epigenetic alterations in CRC, exploring their implications for diagnosis, prevention, and treatment. By integrating multi-omics approaches, single-cell technologies, and model organism studies, future research can enhance the specificity and efficacy of epigenetic-based therapies. Shortly, a combination of advanced gene-editing technologies, targeted epidrugs, and lifestyle interventions may pave the way for more effective and personalized CRC treatment strategies.},
}

Humans
*Colorectal Neoplasms/genetics/therapy/pathology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Epigenesis, Genetic
Animals
DNA Methylation
Gene Expression Regulation, Neoplastic
Epigenome Editing

@article {pmid39568260,
year = {2025},
author = {Wang, B and Fan, A and Liu, M and Zhou, Y and Zhang, W and Yan, J},
title = {An Integrated Rapid Detection of Botryosphaeriaceae Species in Grapevine Based on Recombinase Polymerase Amplification, CRISPR/Cas12a, and Lateral Flow Dipstick.},
journal = {Plant disease},
volume = {109},
number = {5},
pages = {1102-1110},
doi = {10.1094/PDIS-08-24-1615-RE},
pmid = {39568260},
issn = {0191-2917},
mesh = {*Vitis/microbiology ; *Ascomycota/genetics/isolation & purification ; *Plant Diseases/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/genetics/metabolism ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Grapevine Botryosphaeria dieback (GBD), caused by Botryosphaeriaceae species, is an important grapevine trunk disease that poses a threat to grape yield and quality in global viticultural regions. Pathogen diagnosis at the species level using morphological methods is difficult and time-consuming. Therefore, this study aimed to develop a rapid and accurate detection method for the pathogens causing GBD. Recombinase polymerase amplification (RPA) with CRISPR/Cas12a cleavage was combined for detecting pathogens associated with GBD, and lateral flow dipsticks were employed to monitor the outcomes. Based on the β-tubulin sequences of Botryosphaeriaceae and their related species, specific RPA primers and CRISPR/Cas12a CrRNA were designed and subsequently selected for specifically detecting pathogens associated with GBD. Under optimized reaction conditions and systems, the developed RPA/CRISPR-Cas12a detection system specifically detected Botryosphaeriaceae species within 30 min of RPA and 25 min of CRISPR/Cas12a reactions at 37°C. Specificity tests showed that specific fragments were amplified with the RPA primers in the DNA of six Botryosphaeriaceae species found in China, while none of the fragments were amplified in the other 22 nontarget fungal pathogen species of grapevine. The detection sensitivity of this method was 1 pg/μl, which is equal to that of real-time PCR. In summary, our method is simple to perform, produces visual results, does not rely on expensive equipment, and therefore possesses high practical value, providing an efficient and robust detection platform to accelerate the field detection of pathogens associated with GBD.},
}

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

@article {pmid40415627,
year = {2025},
author = {Li, H and Zhu, Q and Zhu, J and Min, J},
title = {[Research progress in the developmental process of non-viral CAR-T technology].},
journal = {Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology},
volume = {41},
number = {5},
pages = {461-467},
pmid = {40415627},
issn = {1007-8738},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Animals ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Gene Editing ; CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; *T-Lymphocytes/immunology ; Neoplasms/therapy/immunology ; },
abstract = {Chimeric antigen receptor T (CAR-T) lymphocytes are at the forefront of adoptive immunotherapy research, and this technology has significantly advanced the prospects of tumor immunotherapy. CAR-T therapy has demonstrated remarkable efficacy in haematological tumours of lymphoid origin and provided therapeutic possibility for solid tumours. Currently, CAR-T cell preparation predominantly involves transfection of T cells with viral vectors. However, the production of viral vectors is time-consuming, expensive, and the vectors have low loading capacity, along with insertion instability. Consequently, there is a pressing need to develop more convenient and precise non-viral gene delivery methods. This paper reviews the most promising non-viral gene delivery technologies, including CRISPR/Cas9 gene editing, transposon systems such as Sleeping Beauty (SB) and PiggyBac (PB), and mRNA, and anticipates the future development of non-viral vector-based CAR-T therapies.},
}

Humans
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/genetics/immunology
Animals
Gene Transfer Techniques
Genetic Vectors/genetics
Gene Editing
CRISPR-Cas Systems/genetics
DNA Transposable Elements/genetics
*T-Lymphocytes/immunology
Neoplasms/therapy/immunology

@article {pmid40414878,
year = {2025},
author = {Lobel, JH and Ingolia, NT},
title = {Precise measurement of molecular phenotypes with barcode-based CRISPRi systems.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {142},
pmid = {40414878},
issn = {1474-760X},
support = {F32 GM148044/GM/NIGMS NIH HHS/United States ; R01 GM135233/GM/NIGMS NIH HHS/United States ; R21 HG012991/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Phenotype ; Humans ; Promoter Regions, Genetic ; },
abstract = {Genome-wide CRISPR-Cas9 screens have untangled regulatory networks driving diverse biological processes. Their success relies on interrogating specific molecular phenotypes and distinguishing key regulators from background effects. Here, we realize these goals by optimizing CRISPR interference with barcoded expression reporter sequencing (CiBER-seq) to dramatically improve the sensitivity and scope of genome-wide screens. We systematically address technical factors that distort phenotypic measurements by normalizing expression reporters against closely matched promoters. We use our improved CiBER-seq to accurately capture known components of well-studied RNA and protein quality control systems. These results demonstrate the precision and versatility of CiBER-seq for dissecting cellular pathways.},
}

*CRISPR-Cas Systems
Phenotype
Humans
Promoter Regions, Genetic

@article {pmid40414010,
year = {2025},
author = {Cao, X and Gao, Z and Yin, P and Wang, H and Yang, L},
title = {Crystal structure and inhibition mechanism of AcrIIA11.},
journal = {Biochemical and biophysical research communications},
volume = {772},
number = {},
pages = {152073},
doi = {10.1016/j.bbrc.2025.152073},
pmid = {40414010},
issn = {1090-2104},
abstract = {Anti-CRISPR (Acr) proteins are naturally evolved inhibitors that precisely target and suppress CRISPR-Cas systems, representing a sophisticated molecular arms race between bacteriophages and their bacterial hosts. While Class 1 systems dominate among sequenced prokaryotic genomes, Class 2 systems remain primary sources of editing tools. Here, we report the structural and mechanistic characterization of AcrIIA11, an anti-CRISPR protein that simultaneously inhibits Streptococcus pyogenes (SpyCas9) and Staphylococcus aureus Cas9 (SauCas9). The 3.2 Å crystal structure reveals a compact α/β fold with distinct electropositive clefts implicated in DNA binding. While DALI analysis identified structural homology to transcriptional regulators and the RecA inhibitor PsiB (RMSD 3.3 Å), functional studies established that AcrIIA11 forms stable ternary complexes with both Cas9 orthologs and sgRNA. Biochemical assays demonstrated stronger inhibition of SauCas9 compared to SpyCas9, with EMSA revealing a critical dichotomy: AcrIIA11 maintains SauCas9-sgRNA binding to specific target DNA while completely blocking cleavage activity. Computational docking localizes AcrIIA11 at the HNH-RuvC interface without obstructing DNA-binding channels in SauCas9, suggesting allosteric inhibition through HNH domain displacement. This work establishes AcrIIA11 as a dual-purpose Cas9 inhibitor that preserves target recognition while inactivating nuclease function-a mechanism with potential applications in precision CRISPR control.},
}

@article {pmid40413996,
year = {2025},
author = {Duan, M and Li, G and Shen, J and Dai, R and Li, X and Liu, Z and Jia, F},
title = {A CRISPR/Cas12a biosensor for portable and accessible detection of Salmonella typhimurium via multi-indicator pH millidisc colorimetry and smartphone imaging platform.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117611},
doi = {10.1016/j.bios.2025.117611},
pmid = {40413996},
issn = {1873-4235},
abstract = {Conventional colorimetric CRISPR/Cas methods rely on a single chromogenic substrate and bulky and specialized signal detection instrument, which hinder their practical application. Herein, a portable and accessible CRISPR/Cas12a biosensor was for the first time reported to sensitively quantify Salmonella enterica serovar typhimurium (S. typhimurium), utilizing a multi-indicator pH millimeter disc (millidisc) for signal visualization, combined with a smartphone-based imaging platform for signal readout. The pH millidisc, composed of multiple indicators, possessed sensitive pH responsiveness and exhibited diverse color changes. The self-developed RGB mini-program, named DeepFood, was designed for portable smartphone use, featuring user-friendly operation and trend visualization for preliminary result analysis. Based on the RGB signal variation pattern with S. typhimurium, a distinct Senh signal type was designed, enhancing the signal-to-noise ratio from 3.38 to 7.11. Compared to the R signal type, the Senh signal type improved detection sensitivity by 36.23-fold (7.26 CFU/mL) in 0.01 M PBS buffer and 15.53-fold (1.41 × 10[2] CFU/mL) in chicken. The proposed biosensor offers significant improvements in detection sensitivity and practical applicability, with potential in food safety and environmental protection.},
}

@article {pmid40413357,
year = {2025},
author = {Jiang, B and An, Z and Niu, L and Qin, D},
title = {Precise genome editing process and its applications in plants driven by AI.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {109},
pmid = {40413357},
issn = {1438-7948},
support = {BLX202316//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genome, Plant ; *Plants/genetics ; *Artificial Intelligence ; Plant Breeding ; },
abstract = {Genome editing technologies have emerged as the keystone of biotechnological research, enabling precise gene modification. The field has evolved rapidly through revolutionary advancements, transitioning from early explorations to the breakthrough of the CRISPR-Cas system. The emergence of the CRISPR-Cas system represents a huge leap in genome editing, prompting the development of advanced tools such as base and prime editors, thereby enhancing precise genomic engineering capabilities. The rapid integration of AI across disciplines is now driving another transformative phase in genome editing, streamlining workflows and enhancing precision. The application prospects of genome editing technology are extensive, particularly in plant breeding, where it has already presented unparalleled opportunities for improving plant traits. Here, we review early genome editing technologies, including meganucleases, ZFNs, TALENs, and CRISPR-Cas systems. We also provide a detailed introduction to next-generation editing tools-such as base editors and prime editors-and their latest applications in plants. At the same time, we summarize and prospect the cutting-edge developments and future trends of genome editing technologies in combination with the rapidly rising AI technology, including optimizing editing systems, predicting the efficiency of editing sites and designing editing strategies. We are convinced that as these technologies progress and their utilization expands, they will provide pioneering solutions to global challenges, ushering in an era of health, prosperity, and sustainability.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genome, Plant
*Plants/genetics
*Artificial Intelligence
Plant Breeding

@article {pmid40316115,
year = {2025},
author = {Tao, Z and Tian, C and Zhong, C and Ji, B and Li, W and Zhao, Y},
title = {The role of NhaA protein in modulating antibiotic tolerance in Escherichia coli.},
journal = {International journal of biological macromolecules},
volume = {311},
number = {Pt 2},
pages = {143721},
doi = {10.1016/j.ijbiomac.2025.143721},
pmid = {40316115},
issn = {1879-0003},
mesh = {*Escherichia coli/drug effects/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; *Escherichia coli Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics ; Adenosine Triphosphate/metabolism ; Proton-Motive Force/drug effects ; Cell Membrane Permeability/drug effects ; Membrane Fluidity/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; },
abstract = {As microbial resistance and recurrent bacterial infections escalate, the growing understanding of the interplay between antibiotic resistance and tolerance has sparked significant interest in the latter. Previous studies have demonstrated that the deletion of cation/proton antiporters (CPAs) induces bacterial phenotypes, such as slow growth and prolonged lag phases, which contribute to the development of tolerance. This study investigates the role of the NhaA protein in antibiotic tolerance in Escherichia coli using CRISPR/Cas9 gene editing to delete the NhaA protein. Our results suggest that the NhaA protein plays a key role in modulating antibiotic tolerance. In response to NhaA deletion, E. coli adapts through multiple mechanisms, including changes in membrane permeability, enhanced efflux activity, increased membrane fluidity, disruption of the proton motive force (PMF), and a reduction in intracellular ATP levels. These adaptive changes collectively promote the development of antibiotic tolerance. Understanding these tolerance mechanisms could uncover new therapeutic targets, help prevent the emergence of tolerance, or sustain bacteria cells in a tolerant state, providing crucial strategies to combat the rise of antibiotic-resistant bacteria.},
}

*Escherichia coli/drug effects/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
*Escherichia coli Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics
Adenosine Triphosphate/metabolism
Proton-Motive Force/drug effects
Cell Membrane Permeability/drug effects
Membrane Fluidity/drug effects
Microbial Sensitivity Tests
CRISPR-Cas Systems

@article {pmid40158793,
year = {2025},
author = {Sheng, T and Meng, XZ and Yu, Q and Lv, W and Chen, Y and Cong, Q and Li, W and Gui, L and Li, J and Xu, X},
title = {Transcriptome analysis and CRISPR-Cas9-mediated mutagenesis identify gpr116 as a candidate gene for growth promotion in grass carp (Ctenopharyngodon idella).},
journal = {Comparative biochemistry and physiology. Part A, Molecular & integrative physiology},
volume = {305},
number = {},
pages = {111850},
doi = {10.1016/j.cbpa.2025.111850},
pmid = {40158793},
issn = {1531-4332},
mesh = {Animals ; *Carps/genetics/growth & development ; *CRISPR-Cas Systems ; Zebrafish/genetics/growth & development ; *Receptors, G-Protein-Coupled/genetics/metabolism ; Gene Expression Profiling ; *Fish Proteins/genetics/metabolism ; *Transcriptome ; Mutagenesis ; Brain/metabolism ; },
abstract = {Grass carp (Ctenopharyngodon idella) is an economically important aquaculture species known for its considerable variability in growth performance. In this study, we investigated the growth phenotype by comparing fast-growing and slow-growing groups. Microstructural analyses revealed that slow-growing fish exhibited significantly larger myofibrillar gaps and lower muscle fiber density. To elucidate the underlying molecular basis, we performed transcriptome (RNA-Seq) analysis of brain and dorsal muscle tissues. 328 differentially expressed genes (DEGs) were identified in dorsal muscle tissue (33 up and 295 down-regulated) and 228 in brain tissue (17 up and 211 down-regulated). Gene Ontology and KEGG enrichment analyses indicated that the DEGs were closely associated with apoptosis and angiogenesis pathways. Among the candidate genes, gpr116 was significantly up-regulated in the brain and dorsal muscle tissue of the fast-growing group. Finally, CRISPR-Cas9-mediated knockout in a zebrafish model confirmed that gpr116 deletion significantly restricted growth, underscoring its pivotal role in the growth regulation of grass carp. These discoveries lay significant groundwork for deeper exploration of growth regulation mechanisms in grass carp and offer important clues for selective breeding of key growth marker genes in this species.},
}

Animals
*Carps/genetics/growth & development
*CRISPR-Cas Systems
Zebrafish/genetics/growth & development
*Receptors, G-Protein-Coupled/genetics/metabolism
Gene Expression Profiling
*Fish Proteins/genetics/metabolism
*Transcriptome
Mutagenesis
Brain/metabolism

@article {pmid40147495,
year = {2025},
author = {Panting, M and Holme, IB and Dionisio, G and Brinch-Pedersen, H},
title = {Simplex and multiplex CRISPR/Cas9-mediated knockout of grain protease inhibitors in model and commercial barley improves hydrolysis of barley and soy storage proteins.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2418-2428},
doi = {10.1111/pbi.70065},
pmid = {40147495},
issn = {1467-7652},
support = {8055-00038B//Innovationsfonden/ ; NNF19OC005658//Novo Nordisk Fonden/ ; 101060057//Horizon Europe/ ; },
mesh = {*Hordeum/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Protease Inhibitors/metabolism ; *Soybean Proteins/metabolism ; *Seed Storage Proteins/metabolism ; *Plant Proteins/metabolism/genetics ; Hydrolysis ; Edible Grain/genetics/metabolism ; Seeds/metabolism/genetics ; Gene Knockout Techniques ; Glycine max/metabolism ; Proteolysis ; },
abstract = {Anti-nutritional factors in plant seeds diminish the utilization of nutrients in feed and food. Among these, protease inhibitors inhibit protein degradation by exogenous proteases during digestion. Through conventional and selection-gene-free genome editing using ovules as explants, we used simplex and multiplex CRISPR/Cas9 for studying the impact of chymotrypsin inhibitor CI-1A, CI-1B and CI-2, Bowman-Birk trypsin inhibitor, Serpin-Z4, and barley ɑ-amylase/subtilisin inhibitor on barley and soybean storage protein degradation. Mutants were generated in the commercial cultivar Stairway, having a high level of protease inhibition, and the barley model cultivar Golden Promise, having a lower inhibition level. In Golden Promise, all individual knockouts decreased the inhibition of the three proteases α-chymotrypsin, trypsin and the commercial feed protease Ronozyme ProAct significantly. The triple knockout of all chymotrypsin inhibitors further decreased the inhibition of α-chymotrypsin and Ronozyme ProAct proteases. Degradations of recombinant barley storage proteins B- and C-hordeins were significantly improved following mutagenesis. In Stairway, a single knockout of CI-1A almost compares to the effect on the proteases achieved for the triple knockout in Golden promise, uncovering CI-1A as the major protease inhibitor in that cultivar. The Stairway mutant demonstrated significantly improved degradation of recombinant barley hordeins and in the soybean storage proteins glycinin and β-conglycinin. The results of this study provide insights into cereal protease inhibitor genes and their negative effects on the degradation of barley storage protein and the most important plant protein from soybeans. The study suggests a future focus on plant protease inhibitors as a major target for improving feed and food protein digestibility.},
}

*Hordeum/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Protease Inhibitors/metabolism
*Soybean Proteins/metabolism
*Seed Storage Proteins/metabolism
*Plant Proteins/metabolism/genetics
Hydrolysis
Edible Grain/genetics/metabolism
Seeds/metabolism/genetics
Gene Knockout Techniques
Glycine max/metabolism
Proteolysis

@article {pmid40139718,
year = {2025},
author = {Tian, M and Luo, L and Jin, B and Liu, J and Chen, T and Tang, J and Shen, Y and Zhang, H and Guo, J and Zhang, H and Cui, G and Huang, L},
title = {Highly efficient Agrobacterium rhizogenes-mediated gene editing system in Salvia miltiorrhiza inbred line bh2-7.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2406-2417},
doi = {10.1111/pbi.70029},
pmid = {40139718},
issn = {1467-7652},
support = {CI2021A04109//CACMS Innovation Fund/ ; ZZ14-YQ-049//The Fundamental Research Funds for the Central public welfare research institutes/ ; 2023YFC3503900//National Key Research and Development Program of China/ ; 82274054//National Natural Science Foundation of China/ ; 2060302//Key project at central government level: the ability to establish sustainable use of valuable Chinese Medicine Resources/ ; TSQN202103160//Supported by the Taishan Scholars Program/ ; },
mesh = {*Agrobacterium/genetics ; *Gene Editing/methods ; *Salvia miltiorrhiza/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.},
}

*Agrobacterium/genetics
*Gene Editing/methods
*Salvia miltiorrhiza/genetics
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Genome, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40112038,
year = {2025},
author = {Xu, T and Li, Y and Liu, X and Yang, X and Huang, Z and Xing, J and Liang, C and Li, J and Tan, Y and Zhang, S and Qi, J and Ye, D and Li, Z and Cao, J and Tang, C and Liu, K},
title = {Rubber biosynthesis drives the biogenesis and development of rubber particles, the rubber-producing organelles.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {2303-2316},
doi = {10.1111/pbi.70052},
pmid = {40112038},
issn = {1467-7652},
support = {32201450//National Natural Science Foundation of China/ ; 32160383//National Natural Science Foundation of China/ ; 31825007//National Natural Science Foundation of China/ ; 32101543//National Natural Science Foundation of China/ ; },
mesh = {*Latex/metabolism/biosynthesis ; *Rubber/metabolism ; *Taraxacum/metabolism/genetics ; Plant Proteins/metabolism/genetics ; *Organelles/metabolism ; Transferases/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Rubber particles (RPs) are specialized organelles for the biosynthesis and storage of natural rubber in rubber-producing plants. However, the mechanisms underlying the biogenesis and development of RPs remain unclear. In this study, two latex-specific cis-prenyltransferases (CPTs), TkCPT1 and TkCPT2, were identified in Taraxacum kok-saghyz, with almost identical orthologues retained across other Taraxacum species. For the first time, Tkcpt1 single and Tkcpt1/2 double mutants were successfully generated using the CRISPR/Cas9 system. Rubber biosynthesis was significantly depressed in Tkcpt1 mutants and completely blocked in Tkcpt1/2 mutants. The absence of RPs in the Tkcpt1/2 was confirmed using oil red O and Nile red staining, high-speed centrifugal stratification, cryo-SEM and TEM on fresh latex or laticifer cells. Transcriptomic and proteomic analyses revealed that, in the latex of Tkcpt1/2, rubber biosynthesis was blocked at the protein level, while metabolomic profiling indicated an enrichment of lipids and terpenoids. Furthermore, knockout of TkCPTL1, a latex-specific CPT-like gene that encodes a rubber transferase activator, resulted in outright disruption of rubber biosynthesis and RP ontogeny, a phenotype similar to that of Tkcpt1/2 mutants. These findings indicate that rubber biosynthesis is a driving force for the biogenesis and development of RPs, providing new insights into rubber production mechanisms.},
}

*Latex/metabolism/biosynthesis
*Rubber/metabolism
*Taraxacum/metabolism/genetics
Plant Proteins/metabolism/genetics
*Organelles/metabolism
Transferases/metabolism/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid40029794,
year = {2025},
author = {Livneh, Y and Leor-Librach, E and Agmon, D and Makov-Bouaniche, T and Tiwari, V and Shor, E and Yeselson, Y and Masci, T and Schaffer, A and Charuvi, D and Hirschberg, J and Vainstein, A},
title = {Combined enhancement of ascorbic acid, β-carotene and zeaxanthin in gene-edited lettuce.},
journal = {Plant biotechnology journal},
volume = {23},
number = {6},
pages = {1954-1967},
doi = {10.1111/pbi.70018},
pmid = {40029794},
issn = {1467-7652},
support = {20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; 12-01-0037//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Zeaxanthins/metabolism ; *beta Carotene/metabolism ; *Lactuca/genetics/metabolism ; *Ascorbic Acid/metabolism ; *Gene Editing ; Intramolecular Lyases/genetics/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Lettuce is widely grown and consumed but provides lower nutritional value compared to other leafy greens, particularly in the essential vitamins A and C. To address this, major control points in carotenoid and ascorbic acid (AsA) production were targeted using a viral-based CRISPR/Cas9 system in the commercial lettuce cultivar 'Noga'. Knockout of lycopene ε-cyclase (LCY-ε), the enzymatic gatekeeper opposing production of β-branch carotenoids, increased β-carotene (provitamin A) levels up to 2.7-fold and facilitated zeaxanthin accumulation up to 4.3 μg/g fresh weight. Chlorophyll fluorescence measurements revealed that photosystem II efficiency was unaffected in LCY-ε mutants, though their non-photochemical quenching (NPQ) capacity decreased at light intensities above 400 μmol m[2] s[-1]. However, the gene-edited plants exhibited normal growth and comparable plant mass, despite the absence of two major lettuce xanthophylls, lutein and lactucaxanthin. Modifications in a regulatory region in the upstream ORF of GDP-L-galactose phosphorylase 1 and 2 (uGGP1 and uGGP2), the rate-limiting enzyme in AsA production, resulted in an average 6.9-fold increase in AsA levels. The mutation in uGGP2 was found to dominantly influence AsA over-accumulation. Knockout lines that combined the mutations in LCY-ε, uGGP1, uGGP2 and in carotenoid cleavage dioxygenase 4a (CCD4a), an isozyme involved in β-carotene degradation in lettuce, exhibited significantly enhanced content of AsA, β-carotene and zeaxanthin. Our results demonstrate the potential of multi-pathway gene editing to 'supercharge' economically important crops such as lettuce as a means to address micronutrient deficiencies in modern diets.},
}

*Zeaxanthins/metabolism
*beta Carotene/metabolism
*Lactuca/genetics/metabolism
*Ascorbic Acid/metabolism
*Gene Editing
Intramolecular Lyases/genetics/metabolism
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid39779901,
year = {2025},
author = {Son, HI and Hamrick, GS and Shende, AR and Kim, K and Yang, K and Huang, TJ and You, L},
title = {Population-level amplification of gene regulation by programmable gene transfer.},
journal = {Nature chemical biology},
volume = {21},
number = {6},
pages = {939-948},
pmid = {39779901},
issn = {1552-4469},
support = {R01EB031869//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; *Gene Transfer Techniques ; *Escherichia coli/genetics ; CRISPR-Cas Systems ; },
abstract = {Engineering cells to sense and respond to environmental cues often focuses on maximizing gene regulation at the single-cell level. Inspired by population-level control mechanisms like the immune response, we demonstrate dynamic control and amplification of gene regulation in bacterial populations using programmable plasmid-mediated gene transfer. By regulating plasmid loss rate, transfer rate and fitness effects via Cas9 endonuclease, F conjugation machinery and antibiotic selection, we modulate the fraction of plasmid-carrying cells, serving as an amplification factor for single-cell-level regulation. This approach expands the dynamic range of gene expression and allows orthogonal control across populations. Our platform offers a versatile strategy for dynamically regulating gene expression in engineered microbial communities.},
}

Plasmids/genetics
*Gene Expression Regulation, Bacterial
*Gene Transfer Techniques
*Escherichia coli/genetics
CRISPR-Cas Systems

@article {pmid40412199,
year = {2025},
author = {Shen, Y and Tang, X and Wang, J and Dai, H and Cui, Y and Hu, Q and Wu, Y and Jia, F and Hao, G},
title = {Dual nuclease-amplified sensitive biosensor for enrofloxacin detection using a DNase I-assisted CRISPR/Cas12a (CRISPR-DNase I) system.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128367},
doi = {10.1016/j.talanta.2025.128367},
pmid = {40412199},
issn = {1873-3573},
abstract = {Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.},
}

@article {pmid40411669,
year = {2025},
author = {Hassan, HM and Zubair, A and Helal, MH and Almagharbeh, WT and Elmagzoub, RM},
title = {New hope and promise with CRISPR-Cas9 technology for the treatment of HIV.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {108},
pmid = {40411669},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Humans ; *HIV Infections/therapy/genetics/virology ; *Gene Editing/methods ; *HIV-1/genetics ; Genetic Therapy/methods ; },
abstract = {The commencement of Highly Active Antiretroviral Therapy almost completely stopped viral replication, enabling the immune system to restore its full functionality. The rise in life expectancy has resulted in a decrease in the incidence of classical infections and HIV-associated cancers. HAART has raised concerns, including its exorbitant cost (which hinders its implementation in developing nations), the need for strict adherence, and the potential for both immediate and prolonged ill effects. Lipodystrophy is a significant long-term consequence of HIV that may result in central fat accumulation and severe peripheral fat depletion. Current initiatives to tackle these difficulties include the global expansion of access to HAART, the development of novel drugs that mitigate early side effects, and the introduction of once-daily drug combinations that enhance adherence. The CRISPR-Cas9 system has facilitated the creation of a powerful instrument for precise gene editing. This method has lately established itself as the gold standard for efficient HIV-1 genome editing in HIV therapy, owing to progress in related disciplines. CRISPR may be customized to cleave specific sequences by altering Cas9. This article offers a concise overview of promising CRISPR-Cas9 technology. This technique has the potential to halt the transmission of HIV-1 and alleviate its symptoms. CRISPR-Cas9 technology will be significant in the fight against HIV-1 in the future.},
}

*CRISPR-Cas Systems
Humans
*HIV Infections/therapy/genetics/virology
*Gene Editing/methods
*HIV-1/genetics
Genetic Therapy/methods

@article {pmid40410287,
year = {2025},
author = {Wei, J and Shao, Y and Liang, Y and Bu, X and Zhou, W and Liang, Y and Li, Y},
title = {A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {793},
pmid = {40410287},
issn = {2399-3642},
support = {32170096//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Thermus thermophilus/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Bacterial/metabolism/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {CRISPR-Cas systems are small RNA-guided immune systems in prokaryotes. CRISPR RNA (crRNA) provides sequence specificity and programmability, guiding the effector complex to cleave target nucleic acids. Cas6 family ribonucleases can cleave precursor crRNA to generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, in hosts harboring multiple CRISPR systems, the biological functions of the co-occurrence of various Cas6 proteins and their cross-cleavage activity toward different types of crRNAs remain largely unexplored. In this study, we biochemically characterized the cross-cleavage activity of two Cas6 proteins in Thermus thermophilus HB27 and first found that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of type I Cascade complex. We further demonstrated that the type III-associated Cas6 protein could act as a negative regulator by competing with the I-B Cas6 protein during the assembly of type I-B Cascade complex, significantly suppressing the interference activity of type I-B system. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems.},
}

*Thermus thermophilus/genetics/metabolism/enzymology
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics
RNA, Bacterial/metabolism/genetics
Gene Expression Regulation, Bacterial

@article {pmid40409866,
year = {2025},
author = {Wang, L and Wu, Y and Pan, S and Pan, F and Chen, J},
title = {Rapid and sensitive detection of Karlodinium veneficum using RAA and CRISPR-Cas12a technologies.},
journal = {Harmful algae},
volume = {146},
number = {},
pages = {102864},
doi = {10.1016/j.hal.2025.102864},
pmid = {40409866},
issn = {1878-1470},
mesh = {*Dinoflagellida/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Harmful Algal Bloom ; },
abstract = {The harmful algal species Karlodinium veneficum (K. veneficum) poses a significant threat to aquatic ecosystems, economic stability, and human health due to its toxin production and widespread occurrence. Rapid climatic changes and eutrophication have intensified harmful algal blooms (HABs), making the timely detection of K. veneficum critical. To address this need, we developed a rapid and accurate detection method of K. veneficum by combining Recombinase Aided Amplification (RAA) with CRISPR/LbCas12a. This method targets the internal transcribed spacer (ITS) sequence of K. veneficum and utilizes the "collateral activity" of CRISPR/LbCas12a for visualization. Our method can detect plasmid DNA as low as 5.9 × 10[2] copies/µL and genomic DNA as low as 3.6 × 10[-2] ng/µL, achieving a detection limit of 10 cells of K. veneficum through a simplified DNA extraction process. The entire detection process, from DNA crude extract to result visualization, can be completed in as fast as 90 min, making it suitable for field applications requiring a rapid response. In addition, our method was validated against a wide range of non-target microalgae species, confirming its specificity to K. veneficum and eliminating the risk of cross-reactivity. Overall, the RAA-CRISPR/LbCas12a system is simple, accurate, and sensitive, showing great potential for field applications in monitoring K. veneficum.},
}

*Dinoflagellida/genetics/isolation & purification
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Harmful Algal Bloom

@article {pmid40409549,
year = {2025},
author = {Okada, R and Sakaguchi, R and Komaki, T and Nonaka, R and Polat, OK and Kihara, T and Asanuma, K and Yamamoto, T and Isaka, Y and Mori, Y and Mori, MX},
title = {Delayed inactivation of TRPC6 as a determinative characteristic of FSGS-associated variants.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110256},
doi = {10.1016/j.jbc.2025.110256},
pmid = {40409549},
issn = {1083-351X},
abstract = {Transient receptor potential canonical 6 (TRPC6) is a receptor-operated nonspecific cation channel. To date, more than 30 TRPC6 variants have been reported to focal segmental glomerulosclerosis (FSGS), which can present from infancy to adulthood and is characterized by proteinuria and often nephrotic syndrome leading to kidney failure. These variants may exhibit gain-of-function (e.g. K874X) or loss-of-function (e.g. L395A, G757D) phenotypes, making the role of TRPC6 in FSGS controversial. Here, we characterized Ca[2+]-dependent inactivation (CDI) of TRPC6 after the receptor activation and found that >85% of TRPC6 variants exhibit delayed CDI. Thus, prolonged TRPC6 channel opening due to impaired inactivation may be a common feature of FSGS-associated variants. This effect was confirmed in immortalized mouse podocytes (MPC-5) in which the coiled-coil (CC) domain was deleted from the channel (C6ΔCC). Podocytes expressing C6ΔCC exhibited delayed CDI and increased basal Ca[2+] levels as well as disruption of the F-actin cytoskeleton. Moreover, transcriptomic data from C6ΔCC-expressing podocytes showed weak expression of the podocyte markers Synpo and Magi2. These results indicate that CDI of TRPC6 is critical for maintaining proper podocyte function. Notably, we observed a correlation between the magnitude of the prolongation of TRPC6 channel activity and the age diagnosed with FSGS. Our findings thus demonstrate that delayed inactivation due to lack of CDI is a determinative characteristic of FSGS-associated TRPC6 variants, affecting both the structure and function of glomerular podocytes.},
}

@article {pmid40409480,
year = {2025},
author = {Yi, B and Zhou, B and Zhou, D and Yang, L and Xu, H},
title = {CRISPR/Cas-powered nucleic acid amplification and amplification-free biosensors for public safety detection: Principles, advances and prospects.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108609},
doi = {10.1016/j.biotechadv.2025.108609},
pmid = {40409480},
issn = {1873-1899},
abstract = {Rapid, accurate, cost-effective, and efficient ultrasensitive detection strategies are essential for public health safety (including food safety, disease prevention and environmental governance). The CRISPR/CRISPR-associated (Cas) detection is a cutting-edge technology that has been widely used in the detection of public health safety due to its targeted cleavage properties (signal amplification), attomolar level sensitivity, high specificity (recognizing single-base mismatches), and rapid turnover time. However, the current research about CRISPR/Cas-based biosensors is not clear, such as mechanism problem and application differences of integrating CRISPR/Cas system with other technologies, and how to further innovate and develop in the future. Therefore, further detailed analysis and comparative discussion of CRISPR/Cas-based biosensors is needed. Currently, CRISPR/Cas system powered biosensors can be mainly categorized into two types: CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors. The two biosensors have different characteristics and advantages. This paper first provides an in-depth investigation of the enzymatic mechanism of CRISPR/Cas system at the molecular level. Then, this paper summarizes the principles and recent advances of CRISPR/Cas system powered nucleic acid amplification biosensors and CRISPR/Cas system powered nucleic acid amplification-free biosensors and discusses their integration mechanisms in depth. More, the differences and application-oriented between the two biosensors are further discussed. Finally, the application orientation and future perspectives of the two biosensors are discussed, and unique insights into the future development of CRISPR/Cas system are provided.},
}

@article {pmid40408893,
year = {2025},
author = {Kong, H and Yi, K and Zhu, X and Chen, L and Wang, H and Ju, E and Lv, S and Lao, YH and Shao, D and Xie, X and Cheng, D and Zhang, Y and Tao, Y and Li, M},
title = {Antifouling fusion-mediated diagnostic platform to detect viral DNA-positive extracellular vesicles for in situ blood-based liquid biopsy.},
journal = {Biosensors & bioelectronics},
volume = {286},
number = {},
pages = {117568},
doi = {10.1016/j.bios.2025.117568},
pmid = {40408893},
issn = {1873-4235},
abstract = {In liquid biopsy, extracellular vesicles (EVs) have emerged as promising biomarkers due to their ability to carry protected nucleic acids. In particular, DNA enclosed within these vesicles shows great diagnostic potential for monitoring oncovirus-related disease progression. However, current methods still require labor-intensive procedures and bulk analysis. Additionally, in situ detection from blood is hindered by abundant serum proteins, interfering with the accuracy of diagnosis. To address these limitations, we developed an antifouling fusion-mediated CRISPR/Cas detector (AFFECTOR) as a user-friendly and efficient diagnostic platform for directly detecting EV-contained viral DNA in serum samples. Leveraging zwitterionic phosphatidylcholine to resist protein interference, the platform enables stable membrane fusion with intact EVs even in serum-containing environments, allowing highly specific and sensitive detection of internal DNA via the CRISPR/Cas12a sensing system, lasting just 2 h at 37 °C. In clinical samples from oncovirus-infected patients and healthy donors, the platform achieved one-step detection of viral DNA-positive EVs. Notably, viral DNA in circulating EVs was found for the first time to correlate with oncovirus infection stages. Overall, this platform provides a practical tool for diagnostic applications and expands the detection window in liquid biopsy.},
}

@article {pmid40405306,
year = {2025},
author = {Marei, HE},
title = {Stem cell therapy: a revolutionary cure or a pandora's box.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {255},
pmid = {40405306},
issn = {1757-6512},
mesh = {Humans ; *Stem Cell Transplantation/ethics ; *Stem Cell Research/ethics/legislation & jurisprudence ; Gene Editing ; *Cell- and Tissue-Based Therapy/ethics ; Embryonic Stem Cells ; Animals ; CRISPR-Cas Systems ; },
abstract = {This review article examines how stem cell therapies can cure various diseases and injuries while also discussing the difficulties and moral conundrums that come with their application. The article focuses on the revolutionary developments in stem cell research, especially the introduction of gene editing tools like CRISPR-Cas9, which can potentially improve the safety and effectiveness of stem cell-based treatments. To guarantee the responsible use of stem cells in clinical applications, it is also argued that standardizing clinical procedures and fortifying ethical and regulatory frameworks are essential first steps. The assessment also highlights the substantial obstacles that still need to be addressed, such as the moral dilemmas raised by the use of embryonic stem cells, the dangers of unlicensed stem cell clinics, and the difficulties in obtaining and paying for care for patients. The study emphasizes how critical it is to address these problems to stop exploitation, guarantee patient safety, and increase the accessibility of stem cell therapy. The review also addresses the significance of thorough clinical trials, public education, and policy development to guarantee that stem cell research may fulfill its full potential. The review concludes by describing stem cell research as a promising but complicated topic that necessitates a thorough evaluation of both the hazards and the benefits. To overcome the ethical, legal, and accessibility obstacles and eventually guarantee that stem cell treatments may be safely and fairly included in conventional healthcare, it urges cooperation between the scientific community, legislators, and the general public.},
}

Humans
*Stem Cell Transplantation/ethics
*Stem Cell Research/ethics/legislation & jurisprudence
Gene Editing
*Cell- and Tissue-Based Therapy/ethics
Embryonic Stem Cells
Animals
CRISPR-Cas Systems

@article {pmid40404851,
year = {2025},
author = {Syed, M and Khan, RS and Nazir, S and Khan, S and Ul Islam, Z and Khan, S and Ikuo, N},
title = {Silencing of disease susceptibility genes: an effective disease resistance strategy against fungal pathogens.},
journal = {Plant cell reports},
volume = {44},
number = {6},
pages = {127},
pmid = {40404851},
issn = {1432-203X},
mesh = {*Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Gene Silencing ; *Fungi/pathogenicity/physiology ; RNA Interference ; Host-Pathogen Interactions/genetics ; Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Silencing of target susceptibility (S) genes in plants exhibits a promising and durable strategy for enhanced resistance to fungal pathogens by causing disruption in the host mechanisms that the pathogens exploit, offering an alternative to the traditional resistance gene-based approaches. Devastating fungal diseases have significantly reduced crop productivity, posing a potential threat to global food security. Producing disease-resistant cultivars is the most effective strategy for protecting crops against these fungal pathogens. Typically, susceptibility (S) genes in host plants facilitate the penetration and proliferation of phytopathogens. Perturbation of these S genes can potentially impede the compatibility between the host and the fungal pathogens, thereby providing broad-spectrum and lasting resistance. Consequently, the identification and targeting of S-genes have gained increasing interest in enhancing disease resistance in plants. In this review, we describe three distinct categories of S genes that function during different stages of the infection process. We focus on various gene silencing technologies, including RNA interference (RNAi), virus-induced gene silencing (VIGS), and CRISPR-Cas9, to improve plant disease resistance against fungal pathogens. The numerous examples discussed here illustrate the potential of S-genes for use in plant disease-resistance breeding.},
}

*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Gene Silencing
*Fungi/pathogenicity/physiology
RNA Interference
Host-Pathogen Interactions/genetics
Genes, Plant
CRISPR-Cas Systems

@article {pmid40403646,
year = {2025},
author = {Wu, Y and Kilgore, R and Hetzler, Z and Chu, W and Moore, B and Barbieri, E and Wei, Q and Menegatti, S},
title = {Purification of CRISPR Cas12a from E. coli cell lysates using peptide affinity ligands.},
journal = {Journal of chromatography. A},
volume = {1755},
number = {},
pages = {466065},
doi = {10.1016/j.chroma.2025.466065},
pmid = {40403646},
issn = {1873-3778},
abstract = {CRISPR Cas nucleases are revolutionizing gene therapy by providing a precise and efficient tool for editing the human genome, and are increasingly applied for engineering microorganisms for bioremediation, drought-resistant crops, and livestock with higher productivity. While Cas9 is currently the most widely utilized member of the CRISPR family, Cas12a stands is gaining prominence for its ability to produce staggered cuts in the target DNA while requiring a shorter guide RNA (crRNA). Current methods of Cas purification such as affinity tag, immunoaffinity, and ion exchange chromatography do not provide either the productivity or the purity needed to meet the demand of clinics and biotechnology industries. Responding to this need, this study presents the first affinity ligands for Cas12a purification via affinity chromatography. The ligands were initially designed in silico as peptide mimetics of anti-CRISPR protein inhibitors AcrVA1 and AcrVA4, and ranked experimentally by Cas12a dynamic binding. Selected ligands P5 and P9 were utilized for purifying Cas12a derived from Acidaminococcus sp. (AsCas12a) and Lachnospiraceae sp. (LbCas12a) from clarified Escherichia coli cell lysates. P5-functionalized resin afforded high yield (up to 80 %), purity (> 93 %), and DNA editing activity (∼72 %) of Cas12a from E. coli lysates featuring different Cas12a and host cell protein titers. The characterization of ligand P5 by surface plasmon resonance (SPR) indicated adsorption kinetics (ka ∼ 1.21·10[5] M[-1]s[-1]) and dissociation constant (KD ∼ 1.76·10[-6] M) that confirmed the ligand design criteria and are characteristic of peptide affinity ligands.},
}

@article {pmid40403178,
year = {2025},
author = {Lee, Y and Lee, JH and Lee, T and Shin, M and Yoon, J},
title = {Amplification-Free CRISPR/Cas12a-Based Electrochemical Biosensor with Enhanced Sensitivity for Viral Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00576},
pmid = {40403178},
issn = {2379-3694},
abstract = {To detect contagious viral nucleic acids, traditional biosensors often require target amplification steps or use fluorescence and Raman probes tagged on nucleic acids, which are time-consuming, complex, and expensive. Recently, the CRISPR/Cas12a has received the attraction for development of nucleic acid biosensors, beyond its conventional role-like gene editing, but the enhancement of the sensitivity of CRISPR/Cas-based biosensors is still required to simplify the biosensing steps. Here, we develop a CRISPR/Cas12a-based electrochemical biosensor for the detection of viral nucleic acids in a simple manner. The novel mismatch Ag probe (MAP), as a sensing probe, and the highly conductive gold electrode on indium tin oxide with a nano array (GELITION) are introduced that enable the amplification-free and ultrasensitive detection of nucleic acids using a CRISPR/Cas12a system. The biosensing ability of the developed biosensor is validated using human papillomavirus type 16 and 18 viral DNAs (HPV16 and HPV18), achieving a limit of detection (LOD) of 1 fM without amplification and complex steps. Our developed biosensor is expected to be applicable in detecting various viruses and could contribute to the early detection of future pandemics.},
}

@article {pmid40402294,
year = {2025},
author = {Hassane, AMA and Obiedallah, M and Karimi, J and Khattab, SMR and Hussein, HR and Abo-Dahab, Y and Eltoukhy, A and Abo-Dahab, NF and Abouelela, ME},
title = {Unravelling fungal genome editing revolution: pathological and biotechnological application aspects.},
journal = {Archives of microbiology},
volume = {207},
number = {7},
pages = {150},
pmid = {40402294},
issn = {1432-072X},
mesh = {*Gene Editing/methods ; *Genome, Fungal ; CRISPR-Cas Systems ; *Biotechnology/methods ; *Fungi/genetics/pathogenicity ; Humans ; Candida albicans/genetics/pathogenicity ; Aspergillus fumigatus/genetics/pathogenicity ; },
abstract = {Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.},
}

*Gene Editing/methods
*Genome, Fungal
CRISPR-Cas Systems
*Biotechnology/methods
*Fungi/genetics/pathogenicity
Humans
Candida albicans/genetics/pathogenicity
Aspergillus fumigatus/genetics/pathogenicity

@article {pmid40401996,
year = {2025},
author = {Pandey, N and Misra, C and Rath, D},
title = {Cas11 augments Cascade functions in type I-E CRISPR system but is redundant for gene silencing and plasmid interference.},
journal = {The Biochemical journal},
volume = {},
number = {},
pages = {},
doi = {10.1042/BCJ20253056},
pmid = {40401996},
issn = {1470-8728},
abstract = {The structural and mechanistic complexity of Escherichia coli's type I CRISPR-Cas system compared to the multidomain, single effector protein-based type II systems, limits its application in genome editing and silencing. Despite higher prevalence of the type I endogenous systems in bacteria, significant research has focused on improving the type II systems. While the type-I CRISPR system possesses several advantages over others, it may benefit from further studies to simplify the system for ease of use. To enable this, the dispensability of the type-I Cascade components (Cas8, Cas11, Cas7, Cas5, Cas6) for genome editing and silencing applications was evaluated in vivo. We created deletion variants of each of the Cascade components and investigated their effects on gene silencing and plasmid interference in two genetically distinct Escherichia coli lineages, BW25113, a K-12 strain that bears an endogenous, albeit repressed type I-E CRISPR system and BL21, a natural mutant lacking the type I-E CRISPR-Cascade system. Cas8, Cas7 and Cas5 were found to be indispensable for gene silencing and plasmid interference. Dispensability of Cas6, which is involved in crRNA maturation, was strain-dependent. Notably, Cas11 which has no definitive function assigned to it, was found to be dispensable for gene silencing and plasmid interference.},
}

@article {pmid40401794,
year = {2025},
author = {Jiao, L and Zhou, Q and Sun, D},
title = {CRISPR-Based Regulation for High-Throughput Screening.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00076},
pmid = {40401794},
issn = {2161-5063},
abstract = {CRISPR technology has revolutionized genome editing by enabling precise, permanent modifications to genetic material. To circumvent the irreversible alterations associated with traditional CRISPR methods and facilitate research on both essential and nonessential genes, CRISPR interference or inhibition (CRISPRi) and CRISPR activation (CRISPRa) were developed. The gene-silencing approach leverages an inactivated Cas effector protein paired with guide RNA to obstruct transcription initiation or elongation, while the gene-activation approach exploits the programmability of CRISPR to activate gene expression. Recent advances in CRISPRi technology, in combination with other technologies (e.g., biosensing, sequencing), have significantly expanded its applications, allowing for genome-wide high-throughput screening (HTS) to identify genetic determinants of phenotypes. These screening strategies have been applied in biomedicine, industry, and basic research. This review explores the CRISPR regulation mechanisms, offers an overview of the workflow for genome-wide CRISPR-based regulation for screens, and highlights its superior suitability for HTS across biomedical and industrial applications. Finally, we discuss the limitations of current CRISPRi/a HTS screening methods and envision future directions in CRISPR-mediated HTS research, considering its potential for broader application across diverse fields.},
}

@article {pmid40401239,
year = {2025},
author = {Mak, JK and Bendandi, A and Salim, JA and Mazoni, I and de Moraes, FR and Borro, L and Störtz, F and Rocchia, W and Neshich, G and Minary, P},
title = {Learning to utilize internal protein 3D nanoenvironment descriptors in predicting CRISPR-Cas9 off-target activity.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {2},
pages = {lqaf054},
pmid = {40401239},
issn = {2631-9268},
mesh = {*CRISPR-Cas Systems ; *Machine Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Gene Editing ; Mutation ; },
abstract = {Despite advances in determining the factors influencing cleavage activity of a CRISPR-Cas9 single guide RNA (sgRNA) at an (off-)target DNA sequence, a comprehensive assessment of pertinent physico-chemical/structural descriptors is missing. In particular, studies have not yet directly exploited the information-rich internal protein 3D nanoenvironment of the sgRNA-(off-)target strand DNA pair, which we obtain by harvesting 634 980 residue-level features for CRISPR-Cas9 complexes. As a proof-of-concept study, we simulated the internal protein 3D nanoenvironment for all experimentally available single-base protospacer-adjacent motif-distal mutations for a given sgRNA-target strand pair. By determining the most relevant residue-level features for CRISPR-Cas9 off-target cleavage activity, we developed STING_CRISPR, a machine learning model delivering accurate predictive performance of off-target cleavage activity for the type of single-base mutations considered in this study. By interpreting STING_CRISPR, we identified four important Cas9 residue spatial hotspots and associated structural/physico-chemical descriptor classes influencing CRISPR-Cas9 (off-)target cleavage activity for the sgRNA-target strand pairs covered in this study.},
}

*CRISPR-Cas Systems
*Machine Learning
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
Gene Editing
Mutation

@article {pmid40400348,
year = {2025},
author = {Khatri, M and Shanmugam, NRS and Zhang, X and Patel, RSKR and Yin, Y},
title = {AcrDB update: Predicted 3D structures of anti-CRISPRs in human gut viromes.},
journal = {Protein science : a publication of the Protein Society},
volume = {34},
number = {6},
pages = {e70177},
pmid = {40400348},
issn = {1469-896X},
support = {R01GM140370/NH/NIH HHS/United States ; R21AI171952/NH/NIH HHS/United States ; 58-8042-9-089//U.S. Department of Agriculture (USDA)/ ; },
mesh = {Humans ; *Bacteriophages/chemistry/genetics ; Protein Conformation ; Databases, Protein ; *Viral Proteins/chemistry/genetics/metabolism ; *Gastrointestinal Microbiome ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Models, Molecular ; },
abstract = {Anti-CRISPR (Acr) proteins play a key role in phage-host interactions and hold great promise for advancing genome-editing technologies. However, finding new Acrs has been challenging due to their low sequence similarity. Recent advances in protein structure prediction have opened new pathways for Acr discovery by using 3D structure similarity. This study presents an updated AcrDB, with the following new features not available in other databases: (1) predicted Acrs from human gut virome databases, (2) Acr structures predicted by AlphaFold2, (3) a structural similarity search function to allow users to submit new sequences and structures to search against 3D structures of experimentally known Acrs. The updated AcrDB contains predicted 3D structures of 795 candidate Acrs with structural similarity (TM-score ≥0.7) to known Acrs supported by at least two of the three non-sequence similarity-based tools (TM-Vec, Foldseek, AcrPred). Among these candidate Acrs, 121 are supported by all three tools. AcrDB also includes 3D structures of 122 experimentally characterized Acr proteins. The 121 most confident candidate Acrs were combined with the 122 known Acrs and clustered into 163 sequence similarity-based Acr families. The 163 families were further subject to a structure similarity-based hierarchical clustering, revealing structural similarity between 44 candidate Acr (cAcr) families and 119 known Acr families. The bacterial hosts of these 163 Acr families are mainly from Bacillota, Pseudomonadota, and Bacteroidota, which are all dominant gut bacterial phyla. Many of these 163 Acr families are also co-localized in Acr operons. All the data and visualization are provided on our website: https://pro.unl.edu/AcrDB.},
}

Humans
*Bacteriophages/chemistry/genetics
Protein Conformation
Databases, Protein
*Viral Proteins/chemistry/genetics/metabolism
*Gastrointestinal Microbiome
*Clustered Regularly Interspaced Short Palindromic Repeats
Models, Molecular

@article {pmid40288547,
year = {2025},
author = {Goméz-Quintero, OS and Morales-Moreno, MD and Valdés-Galindo, EG and Cárdenas-Guerra, RE and Hernandez-Garcia, A},
title = {Enhanced production of functional CRISPR-AsCas12a protein in Escherichia coli.},
journal = {Protein expression and purification},
volume = {232},
number = {},
pages = {106722},
doi = {10.1016/j.pep.2025.106722},
pmid = {40288547},
issn = {1096-0279},
mesh = {*Escherichia coli/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis/isolation & purification ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/biosynthesis/isolation & purification ; *Bacterial Proteins/genetics/biosynthesis/isolation & purification ; *Acidaminococcus/genetics/enzymology ; *Endodeoxyribonucleases/genetics/biosynthesis/isolation & purification ; Gene Editing ; },
abstract = {The CRISPR-Cas12a system is a groundbreaking tool widely used for genome editing and diagnostics in biotechnology and biomedicine research laboratories. Despite its growing application, studies optimizing Cas12a protein production at the laboratory scale using straightforward protocols remains scarce. This study aimed to enhance the lab-scale recombinant production of Acidaminococcus sp Cas12a protein (AsCas12a) in E. coli. Through targeted adjustments of simple parameters, AsCas12a production was significantly increased. The optimized conditions included the use of E. coli BL21(DE3), TB medium supplemented with 1 % glucose, induction with 0.3 mM IPTG for at least 6-9 h, and incubation at 30 °C. Notably, these conditions differ from conventional protocols typically used for Cas12a and related proteins, such as Streptococcus pyogenes Cas9. Upon combining all optimized parameters, AsCas12a production increased approximately 3-fold, from 0.95 mg/mL of bacterial lysate under non-optimized conditions to 3.73 mg/mL under optimized ones. After chromatographic purification, the final protein yield rose approximately 4.5-fold, from 5.2 to 23.4 mg/L of culture volume, without compromising functional activity. The purified AsCas12a retained full activity for programmable in vitro DNA cis-cleavage and collateral trans-cleavage, which was successfully applied to detect the N gene of SARS-CoV-2. This optimized method provide an efficient and high-yield approach for producing functional AsCas12a protein using accessible materials and conditions available to many research laboratories worldwide.},
}

*Escherichia coli/genetics/metabolism
Recombinant Proteins/genetics/biosynthesis/isolation & purification
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics/biosynthesis/isolation & purification
*Bacterial Proteins/genetics/biosynthesis/isolation & purification
*Acidaminococcus/genetics/enzymology
*Endodeoxyribonucleases/genetics/biosynthesis/isolation & purification
Gene Editing

@article {pmid40254211,
year = {2025},
author = {Wei, J and Chen, F and Lu, X and Fan, J and Li, M and Huang, J and Liu, N and Zhang, J and Chai, Z and Lu, S},
title = {In silico identification and experimental validation of long-range allosteric inhibition of Staphylococcus aureus Cas9 catalytic activity by an anti-CRISPR protein AcrIIA14.},
journal = {International journal of biological macromolecules},
volume = {310},
number = {Pt 2},
pages = {143324},
doi = {10.1016/j.ijbiomac.2025.143324},
pmid = {40254211},
issn = {1879-0003},
mesh = {*Staphylococcus aureus/enzymology/genetics ; Allosteric Regulation ; Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/antagonists & inhibitors/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/genetics/metabolism/antagonists & inhibitors ; *CRISPR-Cas Systems ; Allosteric Site ; Mutagenesis, Site-Directed ; Computer Simulation ; },
abstract = {Effective temporal and spatial regulation of CRISPR-Cas9 catalytic activity remains a key challenge, limiting the clinical application of CRISPR-Cas9 gene-editing. Here, we investigated the long-range allosteric inhibition of Staphylococcus aureus Cas9 (SauCas9) catalytic activity by its anti-CRISPR (Acr) protein, AcrIIA14, aiming to uncover remote allosteric mechanisms in large protein complexes and identify potential allosteric sites for the design of SauCas9 inhibitors. Through a combined computational-experimental framework integrating extensive molecular dynamics simulations, Markov state models, network community modeling, and site-directed mutagenesis, we identified canonical and non-canonical inhibitory states of SauCas9 regulated by AcrIIA14. Key domains, including REC, L1, HNH, L2, and PI, play crucial roles in transmitting the AcrIIA14-meidated inhibitory signal. Introducing point mutations on the routes of allosteric communication and analyzing these mutants using in vitro DNA cleavage assays and surface plasmon resonance analysis revealed that SauCas9 escaped AcrIIA14's inhibition owing to the disruption of AcrIIA14-meidated allosteric communication. Moreover, two cryptic allosteric sites on SauCas9 were identified as mutations of these sites prevented inhibition of SauCas9 by AcrIIA14. Overall, our results provide a dynamic understanding of CRISPR-Cas9 regulation and an avenue to design SauCas9 inhibitors with a broad range of applications in Cas9 enzyme catalysis, biophysics, and gene-editing.},
}

*Staphylococcus aureus/enzymology/genetics
Allosteric Regulation
Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/antagonists & inhibitors/chemistry/metabolism/genetics
*Bacterial Proteins/chemistry/genetics/metabolism/antagonists & inhibitors
*CRISPR-Cas Systems
Allosteric Site
Mutagenesis, Site-Directed
Computer Simulation

@article {pmid40400168,
year = {2025},
author = {Jiang, M and Zhao, X and Zhang, C and Liu, R and Hu, J and Lv, Y},
title = {Thermus thermophilus Argonaute-Mediated Single Particle Counting Platform for Multiplex Cancer-Related Biomarkers Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02118},
pmid = {40400168},
issn = {1520-6882},
abstract = {The clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) system has achieved remarkable success in the field of nucleic acid detection, while its Achilles' heel lies in the difficulties encountered in flexibility regarding the multiplex detection. As a sister system of CRISPR-Cas, prokaryotic Argonautes (pAgos) have precise recognition, multiturnover, and more importantly multiple specific cleavage characteristics, which is a potential candidate for the next generation of multiplex detection. Herein, a single particle counting platform was developed for the simultaneous detection of three colorectal cancer-related miRNAs (miR-141, miR-31, and miR-21) by combining single particle inductively coupled plasma mass spectrometry (SP-ICPMS) with the Thermus thermophilus Argonaute protein (TtAgo), with nanoparticles as signal probes for cleavage. The platform demonstrated high sensitivity (aM level) and specificity due to the dual-cycle mechanism of exponential isothermal amplification (EXPAR) and TtAgo cleavage, as well as the combination of TtAgo's specific cleavage capability and the multiplex detection advantages of metal stable isotope tagging. Additionally, the platform showed good robustness in human serum and cell extracts, indicating significant potential in clinical applications.},
}

@article {pmid40400140,
year = {2025},
author = {Jordt, LM and Gellert, M and Zelms, F and Bekeschus, S and Lillig, CH},
title = {The thioredoxin-like and one glutaredoxin domain are required to rescue the iron-starvation phenotype of HeLa GLRX3 knock out cells.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70072},
pmid = {40400140},
issn = {1873-3468},
support = {Li984/3-1//Deutsche Forschungsgemeinschaft/ ; Li984/4-1//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Glutaredoxin 3 (Grx3) is a multidomain protein (Trx-GrxA-GrxB) with a Trx-like domain and two Grx domains containing a CGFS motif for binding Fe2S2 clusters. To study the function of these domains, HeLa cells with GLRX3 knockout were generated via CRISPR/Cas. The knockout activated iron-regulatory protein 1, indicating iron starvation due to impaired iron metabolism. Transfection with constructs encoding wild-type or individual domains showed that only the Trx-GrxA construct could rescue the phenotype, matching the effect of full-length Grx3. The specific role of the second Grx domain in human Grx3, absent in simpler eukaryotes such as yeast, remains unclear. While the individual domains are insufficient to rescue the knockout of full-length Grx3, the Trx-GrxA module is functionally critical. Impact statement Glutaredoxin 3 (Grx3) contains a Trx-like domain and two Grx domains. The importance of the domains in higher eukaryotes has not previously been addressed in physiological or cellular contexts. Here, we report GLRX3 knockout results in activation of iron regulatory protein 1, and a Trx-GrxA construct could rescue the phenotype.},
}

@article {pmid40400023,
year = {2025},
author = {Kermanshahi, AZ and Ebrahimi, F and Taherpoor, A and Eslami, N and Baghi, HB},
title = {HPV-driven cancers: a looming threat and the potential of CRISPR/Cas9 for targeted therapy.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {156},
pmid = {40400023},
issn = {1743-422X},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Papillomavirus Infections/therapy/virology/complications ; *Neoplasms/therapy/virology ; *Papillomaviridae/genetics ; *Genetic Therapy/methods ; Oncogene Proteins, Viral/genetics ; Female ; *Uterine Cervical Neoplasms/therapy/virology ; },
abstract = {Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Papillomavirus Infections/therapy/virology/complications
*Neoplasms/therapy/virology
*Papillomaviridae/genetics
*Genetic Therapy/methods
Oncogene Proteins, Viral/genetics
Female
*Uterine Cervical Neoplasms/therapy/virology

@article {pmid40399557,
year = {2025},
author = {Bourgade, B and Xie, H and Lindblad, P and Stensjö, K},
title = {Development of a CRISPR activation system for targeted gene upregulation in Synechocystis sp. PCC 6803.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {772},
pmid = {40399557},
issn = {2399-3642},
support = {2021-01669//Svenska Forskningsrådet Formas (Swedish Research Council Formas)/ ; },
mesh = {*Synechocystis/genetics/metabolism ; *Up-Regulation ; *Gene Expression Regulation, Bacterial ; Metabolic Engineering/methods ; Butanols/metabolism ; Biofuels ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The photosynthetic cyanobacterium Synechocystis sp. PCC 6803 offers a promising sustainable solution for simultaneous CO2 fixation and compound bioproduction. While various heterologous products have now been synthesised in Synechocystis, limited genetic tools hinder further strain engineering for efficient production. Here, we present a versatile CRISPR activation (CRISPRa) system for Synechocystis, enabling robust multiplexed activation of both heterologous and endogenous targets. Following tool characterisation, we applied CRISPRa to explore targets influencing biofuel production, specifically isobutanol (IB) and 3-methyl-1-butanol (3M1B), demonstrating a proof-of-concept approach to identify key reactions constraining compound biosynthesis. Notably, individual upregulation of target genes, such as pyk1, resulted in up to 4-fold increase in IB/3M1B formation while synergetic effects from multiplexed targeting further enhanced compound production, highlighting the value of this tool for rapid metabolic mapping. Interestingly, activation efficacy did not consistently predict increases in compound formation, suggesting complex regulatory interactions influencing bioproduction. This work establishes a CRISPRa system for targeted upregulation in cyanobacteria, providing an adaptable platform for high-throughput screening, metabolic pathway optimisation and functional genomics. Our CRISPRa system provides a crucial advance in the genetic toolbox available for Synechocystis and will facilitate innovative applications in both fundamental research and metabolic engineering in cyanobacteria.},
}

*Synechocystis/genetics/metabolism
*Up-Regulation
*Gene Expression Regulation, Bacterial
Metabolic Engineering/methods
Butanols/metabolism
Biofuels
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40399309,
year = {2025},
author = {Schmerer, N and Janga, H and Aillaud, M and Hoffmann, J and Aznaourova, M and Wende, S and Steding, H and Halder, LD and Uhl, M and Boldt, F and Stiewe, T and Nist, A and Jerrentrup, L and Kirschbaum, A and Ruppert, C and Rossbach, O and Ntini, E and Marsico, A and Valasarajan, C and Backofen, R and Linne, U and Pullamsetti, SS and Schmeck, B and Schulte, LN},
title = {A searchable atlas of pathogen-sensitive lncRNA networks in human macrophages.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4733},
pmid = {40399309},
issn = {2041-1723},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/5/A004/519/06/00.005(0008)/E31//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 10.21.2.024MN//Fritz Thyssen Stiftung (Fritz Thyssen Foundation)/ ; SFB-TR-84 (subproject C10)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; BA 2168/25-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR 167/2 NeuroMac//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CIBSS - EXC-2189 - Project ID 390939984//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-1213 (Project A01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SFB-TR-84 (subproject C1)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; PerMed-COPD (01EK2203A)//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; Deep Legion (031L0288A)//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Macrophages/metabolism/immunology ; *Gene Regulatory Networks ; GATA2 Transcription Factor/genetics/metabolism ; NF-kappa B/metabolism ; Macrophage Activation/genetics ; Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {Long noncoding RNAs (lncRNA) are crucial yet underexplored regulators of human immunity. Here we develop GRADR, a method integrating gradient profiling with RNA-binding proteome analysis, to map the protein interactomes of all expressed RNAs in a single experiment to study mechanisms of lncRNA-mediated regulation of human primary macrophages. Applying GRADR alongside CRISPR-multiomics, we reveal a network of NFκB-dependent lncRNAs, including LINC01215, AC022816.1 and ROCKI, which modulate distinct aspects of macrophage immunity, particularly through interactions with mRNA-processing factors, such as hnRNP proteins. We further uncover the function of ROCKI in repressing the messenger of the anti-inflammatory GATA2 transcription factor, thus promoting macrophage activation. Lastly, all data are consolidated in the SMyLR web interface, a searchable reference catalog for exploring lncRNA functions and pathway-dependencies in immune cells. Our results thus not only highlight the important functions of lncRNAs in immune regulation, but also provide a rich resource for lncRNA studies.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Macrophages/metabolism/immunology
*Gene Regulatory Networks
GATA2 Transcription Factor/genetics/metabolism
NF-kappa B/metabolism
Macrophage Activation/genetics
Gene Expression Regulation
CRISPR-Cas Systems

@article {pmid40399255,
year = {2025},
author = {Riesenberg, S and Kanis, P and Karlic, R and Maricic, T},
title = {Robust prediction of synthetic gRNA activity and cryptic DNA repair by disentangling cellular CRISPR cleavage outcomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4717},
pmid = {40399255},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; INDEL Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; },
abstract = {The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications, as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines, robustly identifies inefficient gRNAs across different published datasets, and is easily accessible via online genome browser tracks. In addition, we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a, which may be relevant for safety in gene therapy applications.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Humans
*DNA Repair/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
INDEL Mutation
CRISPR-Associated Protein 9/metabolism/genetics
DNA Cleavage
CRISPR-Associated Proteins/metabolism/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells
Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins/metabolism/genetics

@article {pmid40397713,
year = {2025},
author = {Teng, H and Hang, Q and Zheng, C and Yan, Y and Liu, S and Zhao, Y and Deng, Y and Nie, L and Wu, W and Sheldon, M and Yu, Z and Shi, W and Gao, J and Meng, C and Martinez, C and Zhang, J and Yao, F and Sun, Y and Zhao, D and Gan, B and Meng, T and Ma, L},
title = {In vivo CRISPR activation screen identifies acyl-CoA-binding protein as a driver of bone metastasis.},
journal = {Science translational medicine},
volume = {17},
number = {799},
pages = {eado7225},
doi = {10.1126/scitranslmed.ado7225},
pmid = {40397713},
issn = {1946-6242},
mesh = {Animals ; *Bone Neoplasms/secondary/genetics/metabolism/pathology ; Humans ; Cell Line, Tumor ; Mice ; *Diazepam Binding Inhibitor/metabolism/genetics ; Lipid Metabolism ; Fatty Acids/metabolism ; Reactive Oxygen Species/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ferroptosis ; Oxidation-Reduction ; Neoplasm Metastasis ; Female ; *CRISPR-Cas Systems/genetics ; },
abstract = {One of the most common sites of cancer metastasis is to the bone. Bone metastasis is associated with substantial morbidity and mortality, and current therapeutic interventions remain largely palliative. Metastasizing tumor cells need to reprogram their metabolic states to adapt to the nutrient environment of distant organs; however, the role and translational relevance of lipid metabolism in bone metastasis remain unclear. Here, we used an in vivo CRISPR activation screening system coupled with positive selection to identify acyl-coenzyme A (CoA) binding protein (ACBP) as a bone metastasis driver. In nonmetastatic and weakly metastatic cancer cells, overexpression of wild-type ACBP, but not the acyl-CoA-binding deficient mutant, stimulated fatty acid oxidation (FAO) and bone metastasis. Conversely, knockout of ACBP in highly bone metastatic cancer cells abrogated metastatic bone colonization. Mechanistically, ACBP-mediated FAO increased ATP and NADPH production, reduced reactive oxygen species, and inhibited lipid peroxidation and ferroptosis. We found that ACBP expression correlated with metabolic signaling, bone metastatic ability, and poor clinical outcomes. In mouse models, pharmacological blockade of FAO or treatment with a ferroptosis inducer inhibited bone metastasis. Together, our findings reveal the role of lipid metabolism in tumor cells adapting and thriving in the bone and identify ACBP as a key regulator of this process. Agents that target FAO or induce ferroptosis represent a promising therapeutic approach for treating bone metastases.},
}

Animals
*Bone Neoplasms/secondary/genetics/metabolism/pathology
Humans
Cell Line, Tumor
Mice
*Diazepam Binding Inhibitor/metabolism/genetics
Lipid Metabolism
Fatty Acids/metabolism
Reactive Oxygen Species/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Ferroptosis
Oxidation-Reduction
Neoplasm Metastasis
Female
*CRISPR-Cas Systems/genetics

@article {pmid40397663,
year = {2025},
author = {Gast, K and Baker, S and Borges, AL and Ward, S and Banfield, JF and Barrangou, R},
title = {Metagenome-Derived CRISPR-Cas12a Mining and Characterization.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0099},
pmid = {40397663},
issn = {2573-1602},
abstract = {The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies has revolutionized genome editing, with continued interest in expanding the CRISPR-associated proteins (Cas) toolbox with diverse, efficient, and specific effectors. CRISPR-Cas12a is a potent, programmable RNA-guided dual nickase, broadly used for genome editing. Here, we mined dairy cow microbial metagenomes for CRISPR-Cas systems, unraveling novel Cas12a enzymes. Using in silico pipelines, we characterized and predicted key drivers of CRISPR-Cas12a activity, encompassing guides and protospacer adjacent motifs for five systems. We next assessed their functional potential in cell-free transcription-translation assays with GFP-based fluorescence readouts. Lastly, we determined their genome editing potential in vivo in Escherichia coli by generating 1 kb knockouts. Unexpectedly, we observed natural sequence variation in the bridge-helix domain of the best-performing candidate and used mutagenesis to alter the activity of Cas12a orthologs, resulting in increased gene editing capabilities of a relatively inefficient candidate. This study illustrates the potential of underexplored metagenomic sequence diversity for the development and refinement of genome editing effectors.},
}

@article {pmid40370093,
year = {2025},
author = {Qiu, S and Chen, L and Zhuang, D and Cao, Y and Wei, R and Cao, X and Chen, Y and Lai, X and Wang, S and Lin, Y and Lin, Z and Zhang, S},
title = {Fluorescence Aptasensor for sST2 Detection Using In Vitro Selected Aptamers.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10910-10918},
doi = {10.1021/acs.analchem.5c01855},
pmid = {40370093},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Interleukin-1 Receptor-Like 1 Protein/blood/analysis ; *Biosensing Techniques/methods ; Limit of Detection ; Fluorescence ; CRISPR-Cas Systems ; },
abstract = {Soluble suppression of tumorigenicity 2 (sST2) is a critical biomarker for heart failure (HF) diagnosis and prognosis, yet conventional antibody-based detection methods suffer from time-consuming protocols and high costs and involve complex detection procedures. To address these challenges, we first screened high-affinity aptamers under clinically relevant conditions and then coupled with the CRISPR/Cas12a system to develop a fluorescence aptasensor for rapid and sensitive sST2 detection. A serum matrix was introduced during aptamer selection to enhance specificity and anti-interference performance in real biological environments. Three sST2-specific aptamers (Apt-1, Apt-2, and Apt-3) were identified with dissociation constants (KD) of 8.42, 46.08, and 25.02 nM, respectively. Among these, Apt-1 demonstrated superior performance, which was utilized to construct a fluorescence biosensor combining aptamer recognition with CRISPR/Cas12a trans-cleavage signal amplification. The sensor achieved a broad linear detection range (5-120 ng/mL) and an ultralow limit of detection (LOD, 0.816 ng/mL) when applied in detecting sST2 in both the buffer and human serum. Notably, the platform exhibited exceptional resistance to interference from HF-related proteins and maintained high accuracy in clinical serum samples, showing a strong correlation (R[2] = 0.9794) with enzyme-linked immunosorbent assay (ELISA) results. By integration of serum-matrix screening and CRISPR-based signal enhancement, this work establishes a robust, cost-effective, and rapid diagnostic tool for sST2 detection.},
}

*Aptamers, Nucleotide/chemistry
Humans
*Interleukin-1 Receptor-Like 1 Protein/blood/analysis
*Biosensing Techniques/methods
Limit of Detection
Fluorescence
CRISPR-Cas Systems

@article {pmid40366179,
year = {2025},
author = {Zhang, Y and Li, W and Chen, S and Zhang, Y and Zhu, Y and Lan, F and Du, H and Fan, R and Zhu, J and Pan, W and Situ, B and Zheng, L and Luo, S and Yan, X},
title = {Layered-Responsive Multivalent Tetrahedral DNA Framework-Decorated CRISPR-Cas12a Nanocapsule Enables Precise and Enhanced Tumor Chemotherapy.},
journal = {ACS nano},
volume = {19},
number = {20},
pages = {19274-19286},
doi = {10.1021/acsnano.5c01747},
pmid = {40366179},
issn = {1936-086X},
mesh = {*Doxorubicin/pharmacology/chemistry/administration & dosage ; Humans ; *DNA/chemistry ; *CRISPR-Cas Systems ; *Nanocapsules/chemistry ; Animals ; Cell Line, Tumor ; Mice ; *Antineoplastic Agents/pharmacology/chemistry ; *Antibiotics, Antineoplastic/pharmacology/chemistry ; Neoplasms/drug therapy ; },
abstract = {The lack of selective tumor targeting and the high toxicity of conventional chemotherapy treatments remain major challenges in cancer therapy. Here, we develop a self-controlled DNA nanostructure-CRISPR-12a system, a triple-locked cascade tumor therapy nanocapsule (Tatna), for efficient and targeted tumor treatment. Tatna integrates structural DNA tetrahedrons (DTs) with high drug-loading capacity, Cas12a/crRNA ribonucleoprotein (Cas12a RNP), and doxorubicin (DOX) to enable multisite response for precise drug delivery and augmented tumor treatment. By incorporation of a nucleolin-targeting aptamer, Tatna achieves selective targeting and efficient tumor cell internalization. Encapsulation in pH-responsive poly l-lactic-co-glycolic acid (PLGA) nanocapsule ensures stable circulation and controlled release of both DOX and Cas12a until tumor-specific activation in the acidic microenvironment. The Cas12a RNP, triggered by APE1 mRNA overexpression in tumor cells, induces trans-cleavage of DTs, releasing DOX and Cas12a to transport into the nucleus and induce enhanced cell apoptosis. This self-regulating and multifunctional approach enhances the efficacy of chemotherapy while reducing off-target effects. Tatna's programmable, tumor-specific delivery system represents a powerful strategy for advancing precision medicine and personalized cancer treatment.},
}

*Doxorubicin/pharmacology/chemistry/administration & dosage
Humans
*DNA/chemistry
*CRISPR-Cas Systems
*Nanocapsules/chemistry
Animals
Cell Line, Tumor
Mice
*Antineoplastic Agents/pharmacology/chemistry
*Antibiotics, Antineoplastic/pharmacology/chemistry
Neoplasms/drug therapy

@article {pmid40360969,
year = {2025},
author = {Dong, J and Ma, W and Zhou, S and Li, X and Deng, L and Hou, C and Huo, D},
title = {Tri-Mode CRISPR-Based Biosensor for miRNA Detection: Enhancing Clinical Diagnostics with Cross-Validation.},
journal = {Analytical chemistry},
volume = {97},
number = {20},
pages = {10938-10946},
doi = {10.1021/acs.analchem.5c02348},
pmid = {40360969},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Electrochemical Techniques ; Metal-Organic Frameworks/chemistry ; Methylene Blue/chemistry ; },
abstract = {In vitro diagnostics require the accurate detection of disease-associated target biomolecules at ultralow concentrations. A multimode sensing strategy is considered as a potential method for in vitro diagnosis because it allows cross-validation of test results through data complementation and self-calibration, and provides double confirmation. Here, we present a CRISPR/Cas12a-powered trimode biosensor (CPTMB) for ultrasensitive and reliable analysis of miRNA. Briefly, the presence of target miRNA initiates rolling circle extension-driven loop-mediated isothermal amplification (R-LAMP), which subsequently activates the trans-cleavage activity of CRISPR/Cas12a. Then, the hairpin probe (HP) biogate on nucleic acid-functionalized MB@Fe-MOF signal probe was degraded by Cas12a, leading to the release of methylene blue (MB) signal molecules encapsulated within Fe-MOF nanocarriers. Due to the capability of MB to generate output responses across three distinct modes: electrochemical (EC), fluorescence (FL), and ultraviolet-visible spectroscopy (UV-vis), a trimodal sensing system is achieved. Benefiting from the efficient signal amplification capabilities of R-LAMP and CRISPR/Cas12a, this strategy enables rapid detection of target miRNA at femtomolar levels within 70 min. Furthermore, the detection results across the three modes cross-validate one another, thereby enhancing the reliability of the analysis. More importantly, the platform has been successfully applied to miRNA analysis in real samples, and the detection results are in good agreement with those of the standard method RT-qPCR, indicating its great potential in the clinical diagnosis of early-stage cancer.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques
Limit of Detection
Electrochemical Techniques
Metal-Organic Frameworks/chemistry
Methylene Blue/chemistry

@article {pmid40323736,
year = {2025},
author = {Chen, Y and Li, Y and Li, P and Li, X and Zhao, S and Zuo, Z},
title = {Catching CRISPR-Cas9 in Action.},
journal = {Journal of chemical theory and computation},
volume = {21},
number = {10},
pages = {5023-5036},
doi = {10.1021/acs.jctc.5c00165},
pmid = {40323736},
issn = {1549-9626},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Quantum Theory ; },
abstract = {CRISPR-Cas9 has revolutionized genome editing, yet its structural dynamics and functional properties remain incompletely understood, partly due to limited atomic-level characterization of its active conformation with a full R-loop. Capitalizing on recent advances in Cas9 structural determination, we constructed a catalytic-state Cas9 model bound to a bona fide R-loop and performed an integrated computational investigation. Our molecular dynamics simulations reveal substantial conformational heterogeneity in the PAM (protospacer-adjacent motif)-distal nontarget DNA strand and adjacent Cas9 regions, leading to dynamically fluctuating interactions, thereby challenging experimental resolution of the full R-loop complex. Comparative analysis highlights a conformational barrier restricting final activation of the HNH nuclease domain, suggesting that strategic modulation of HNH interactions on its two sides could enhance cleavage efficiency. Furthermore, quantum mechanics/molecular mechanics simulations indicate that with H983 protonated at Nε, the RuvC domain favors a phosphate-mediated over a histidine-mediated pathway for nontarget strand cleavage. Additionally, we identify an alternative HNH-mediated target strand cleavage pathway, involving a water nucleophile aligned at the 5' side of the scissile phosphate. Inspired by the basic residue ladder observed in RuvC, we propose extending a similar ladder in HNH to strengthen DNA binding and catalytic activity. Our study provides critical insights into Cas9 structure, dynamics, and catalysis, laying a foundation for the rational design of next-generation CRISPR-Cas9 systems with optimized specificity-efficiency balance.},
}

Molecular Dynamics Simulation
*CRISPR-Cas Systems
DNA/chemistry/metabolism
*CRISPR-Associated Protein 9/chemistry/metabolism
Quantum Theory

@article {pmid40272173,
year = {2025},
author = {Paz, M and Moratorio, G},
title = {Deep mutational scanning and CRISPR-engineered viruses: tools for evolutionary and functional genomics studies.},
journal = {mSphere},
volume = {10},
number = {5},
pages = {e0050824},
doi = {10.1128/msphere.00508-24},
pmid = {40272173},
issn = {2379-5042},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Genomics/methods ; *Genome, Viral ; *Viruses/genetics ; *Evolution, Molecular ; Humans ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Mutational Analysis/methods ; },
abstract = {Recent advancements in synthetic biology and sequencing technologies have revolutionized the ability to manipulate viral genomes with unparalleled precision. This review focuses on two powerful methodologies: deep mutational scanning and CRISPR-based genome editing, that enable comprehensive mutagenesis and detailed functional characterization of viral proteins. These approaches have significantly deepened our understanding of the molecular determinants driving viral evolution and adaptation. Furthermore, we discuss how these advances provide transformative insights for future vaccine development and therapeutic strategies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Genomics/methods
*Genome, Viral
*Viruses/genetics
*Evolution, Molecular
Humans
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Mutational Analysis/methods

@article {pmid40138089,
year = {2025},
author = {Chu, Y and Wei, M and Cao, Z and Chen, L and Tan, J and Bao, W and Yang, F and Zhang, Y and Lin, Y and Zhang, Y and Li, S and Lv, C and Zhou, W and Du, H and Shen, L and Huai, C and Wang, Z and Qin, S},
title = {Integrative analysis based on CRISPR screen identifies apilimod as a potential therapeutic agent for cisplatin-induced acute kidney injury treatment.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1770-1785},
pmid = {40138089},
issn = {1869-1889},
mesh = {*Cisplatin/adverse effects ; *Acute Kidney Injury/chemically induced/drug therapy/genetics ; Animals ; Humans ; Mice ; Apoptosis/drug effects ; *CRISPR-Cas Systems ; *Morpholines/pharmacology/therapeutic use ; Reactive Oxygen Species/metabolism ; Lipid Metabolism/drug effects ; Male ; Antineoplastic Agents/adverse effects ; Signal Transduction/drug effects ; Cell Line ; },
abstract = {Acute kidney injury (AKI), a life-threatening side effect of cisplatin therapy, significantly limits the drug's therapeutic potential. In this study, we conducted a genome-wide CRISPR/Cas9 knockout screen in human renal tubular epithelial cells, integrating the results with transcriptome analyses and the Connectivity Map (CMap) database. Apilimod and elacridar emerged as the top two candidates of mitigating cisplatin-induced nephrotoxicity, with apilimod demonstrating superior efficacy in drug matrix experiments. Apilimod reduced cisplatin-induced apoptosis, inflammation and reactive oxygen species (ROS) generation. Transcriptome analyses suggested that apilimod may protect against cisplatin-induced nephrotoxicity via modulating lipid metabolism. In vitro experiments revealed that apilimod significantly ameliorated cisplatin-induced lipotoxicity by enhancing lipid clearance and upregulating PGC1α-mediated fatty acid oxidation. Mechanism experiments showed that apilimod induces the nuclear translocation of TFEB through the inhibition of its target, PIKfyve, thereby enhancing PGC1α expression and ameliorating lipotoxicity. These protective effects of apilimod were simulated by siRNA-mediated PIKfyve knockdown and diminished by the PGC1α inhibitor SR-18292 and siRNA targeting TFEB, confirming the role of the PIKfyve/TFEB/PGC1α signaling axis in apilimod's renoprotective effects. In vivo, apilimod alleviated apoptosis, inflammation, and lipid accumulation in a cisplatin-induced AKI mouse model. Additionally, apilimod treatment did not compromise the antitumor effect of cisplatin in cancer cells or tumor-bearing mice. Overall, our study suggests that apilimod could be a promising therapeutic agent for the treatment of cisplatin-induced AKI and revealed its underlying molecular mechanism.},
}

*Cisplatin/adverse effects
*Acute Kidney Injury/chemically induced/drug therapy/genetics
Animals
Humans
Mice
Apoptosis/drug effects
*CRISPR-Cas Systems
*Morpholines/pharmacology/therapeutic use
Reactive Oxygen Species/metabolism
Lipid Metabolism/drug effects
Male
Antineoplastic Agents/adverse effects
Signal Transduction/drug effects
Cell Line

@article {pmid40133712,
year = {2025},
author = {Wang, C and Jiang, X and Li, HY and Hu, J and Ji, Q and Wang, Q and Liu, X and Huang, D and Yan, K and Zhao, L and Fan, Y and Wang, S and Ma, S and Belmonte, JCI and Qu, J and Liu, GH and Zhang, W},
title = {RIG-I-driven CDKN1A stabilization reinforces cellular senescence.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1646-1661},
pmid = {40133712},
issn = {1869-1889},
mesh = {*Cellular Senescence/genetics ; Humans ; *Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics ; *DEAD Box Protein 58/metabolism/genetics ; Mesenchymal Stem Cells/metabolism/cytology ; Receptors, Immunologic/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Immunity, Innate ; },
abstract = {The innate immune signaling network follows a canonical format for signal transmission. The innate immune pathway is crucial for defense against pathogens, yet its mechanistic crosstalk with aging processes remains largely unexplored. Retinoic acid-inducible gene-I (RIG-I), a key mediator of antiviral immunity within this pathway, has an enigmatic role in stem cell senescence. Our study reveals that RIG-I levels increase in human genetic and physiological cellular aging models, and its accumulation drives cellular senescence. Conversely, CRISPR/Cas9-mediated RIG-I deletion or pharmacological inhibition in human mesenchymal stem cells (hMSCs) confers resistance to senescence. Mechanistically, RIG-I binds to endogenous mRNAs, with CDKN1A mRNA being a prominent target. Specifically, RIG-I stabilizes CDKN1A mRNA, resulting in elevated CDKN1A transcript levels and increased p21[Cip1] protein expression, which precipitates senescence. Collectively, our findings establish RIG-I as a post-transcriptional regulator of senescence and suggest potential targets for the mitigation of aging-related diseases.},
}

*Cellular Senescence/genetics
Humans
*Cyclin-Dependent Kinase Inhibitor p21/metabolism/genetics
*DEAD Box Protein 58/metabolism/genetics
Mesenchymal Stem Cells/metabolism/cytology
Receptors, Immunologic/metabolism/genetics
RNA, Messenger/metabolism/genetics
CRISPR-Cas Systems
Signal Transduction
Immunity, Innate

@article {pmid39956793,
year = {2025},
author = {Uzun, S and Özcan, Ö and Gök, A and Işık, A and Bakır, S and Günel-Özcan, A and Onbaşılar, İ and Akyol, A},
title = {A new CRISPR-mediated Apc knockout allele leads to pyloric gland adenoma-like gastric polyps in mice with C57BL/6;FVB/N mixed background.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {5},
pages = {922-929},
doi = {10.1002/ame2.70002},
pmid = {39956793},
issn = {2576-2095},
support = {SBAG-215S926//The Scientific and Technological Research Council of Turkey 1001 program/ ; },
mesh = {Animals ; Mice, Inbred C57BL ; Alleles ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Knockout ; Disease Models, Animal ; *Stomach Neoplasms/genetics/pathology ; *Adenomatous Polyposis Coli Protein/genetics ; Male ; *Adenoma/genetics/pathology ; Adenomatous Polyps ; },
abstract = {Adenomatous polyposis coli (APC) mutations are the most frequently identified genetic alteration in sporadic colorectal cancer (CRC) cases, and a myriad of genetically engineered Apc-mutant CRC mouse models have been developed using various genetic manipulation techniques. The advent of the CRISPR/Cas9 system has revolutionized the field of genetic engineering and facilitated the development of new genetically engineered mouse models. In this study, we aimed to develop a novel Apc knockout allele using the CRISPR/Cas9 system and evaluate the phenotypic effects of this new allele in two different mouse strains. For this purpose, exon 16 of mouse Apc gene was targeted with a single-guide RNA, and the mouse carrying an Apc frameshift mutation at codon 750 (Δ750) was chosen as the founder. The mutant FVB-Apc[Δ750] mice were backcrossed with wild-type C57BL/6 mice, and the phenotypic effects of the knockout allele were evaluated in F8-FVB-Apc[Δ750], F4-B6;FVB-Apc[Δ750], and F1-B6;FVB-Apc[Δ750] by a macroscopic and microscopic examination of the gastrointestinal system. The result showed that the mean polyp number was significantly higher in F4-BL6;FVB-Apc[Δ750] than in F8-FVB-Apc[Δ750]. Intestinal polyposis was more prominent in F4-BL6;FVB-Apc[Δ750], whereas a higher number of colon polyps than intestinal polyps were observed in F8-FVB-Apc[Δ750]. Additionally, F1-BL6;FVB-Apc[Δ750] mixed background mice developed gastric polyps that morphologically resembled the pyloric gland adenoma of humans. In conclusion, we developed a novel CRISPR-mediated Apc knockout allele using two mouse strains. We showed that this allele can exert a strain-specific effect on the phenotype of mice and can cause gastric polyp formation.},
}

Animals
Mice, Inbred C57BL
Alleles
Mice
*CRISPR-Cas Systems/genetics
Mice, Knockout
Disease Models, Animal
*Stomach Neoplasms/genetics/pathology
*Adenomatous Polyposis Coli Protein/genetics
Male
*Adenoma/genetics/pathology
Adenomatous Polyps

@article {pmid39821833,
year = {2025},
author = {Li, Y and Fu, Y and Li, Y and Zhang, R and Yang, J and Ma, H and Min, L and Zhang, X},
title = {Reversing anther thermotolerance by manipulating the cis-elements in the promoter of a high-temperature upregulated gene Casein Kinase I in upland cotton.},
journal = {Science China. Life sciences},
volume = {68},
number = {6},
pages = {1558-1569},
pmid = {39821833},
issn = {1869-1889},
mesh = {*Gossypium/genetics/physiology ; *Promoter Regions, Genetic/genetics ; *Thermotolerance/genetics ; Gene Expression Regulation, Plant ; *Flowers/genetics/physiology ; *Casein Kinase I/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Hot Temperature ; Plants, Genetically Modified ; Alleles ; Up-Regulation ; Genotype ; Phenotype ; },
abstract = {High temperature (HT) stress causes male sterility, leading to reduced upland cotton yield. Previously, we identified a key gene, Casein Kinase I (GhCKI), that negatively regulates male fertility in upland cotton under HT. However, conventional genetic manipulations of GhCKI would result in male sterility, hindering its utilization in breeding programs. Here, we engineered quantitative variation for anther thermotolerance-related traits in upland cotton by creating weak promoter alleles of GhCKI genes, using CRISPR/Cas9 and CRISPR/Cpf1 genome editing. Then, we screened and identified two new upland cotton plant lines exhibiting a HT-tolerant phenotype with edited GhCKI promoters, and characterized their corresponding heat-tolerant allelic genotypes. Further research revealed that the primary reason for the HT tolerance of the GhCKI promoter editing mutants is that the trans-acting factors GhMYB73 and GhMYB4, which positively regulate GhCKI expression under HT, failed to bind and activate the expression of GhCKI. Overall, our study not only provides a rapid strategy to generate new beneficial alleles but also offers novel germplasm resources and molecular insights for crop HT tolerance breeding.},
}

*Gossypium/genetics/physiology
*Promoter Regions, Genetic/genetics
*Thermotolerance/genetics
Gene Expression Regulation, Plant
*Flowers/genetics/physiology
*Casein Kinase I/genetics/metabolism
Gene Editing
CRISPR-Cas Systems
Plant Proteins/genetics/metabolism
Hot Temperature
Plants, Genetically Modified
Alleles
Up-Regulation
Genotype
Phenotype

@article {pmid40394594,
year = {2025},
author = {Guo, J and Shan, Y and Hu, G and Zhu, X and Zhao, K and Wu, T and Qiao, Q and Chi, Y and Cui, L and Ge, Y},
title = {Rapid visual detection of Monkeypox virus by one-step LAMP-CRISPR/Cas12b assay.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {151},
pmid = {40394594},
issn = {1743-422X},
support = {2023YFC2605100, 2023YFC2605104,BK20231374, BK20221413//National Key R&D Program of China；Natural science foundation of Jiangsu Province/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Monkeypox virus/isolation & purification/genetics ; Humans ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; },
abstract = {BACKGROUND: Monkeypox virus (MPXV) infection has garnered significant global attention due to its rising incidence and substantial public health implications. A rapid, sensitive, and accurate diagnostic method is urgently required to enable early intervention and effective management of MPXV outbreaks.

RESULTS: In this study, we developed a novel one-step assay that integrates loop-mediated isothermal amplification (LAMP) with CRISPR/Cas12b in one-pot for the detection of MPXV. The entire detection process did not require opening the lid of the reaction tube and could be completed within 40 min using extracted viral nucleic acids, which is faster than real-time quantitative PCR (qPCR). And the results could be interpreted through either real-time fluorescence or naked-eye visualization. The limit of detection (LOD) of the assay was demonstrated to be 6.5 copies per reaction and no cross-reactivity with other pathogens such as HSV, EBV, CVA16, EV-A71, and MV was found. Furthermore, when evaluated using 113 clinical samples, the assay achieved 100% sensitivity (71/71) and 100% specificity (42/42) compared to the qPCR.

CONCLUSIONS: In resource-limited settings, our method requires only a portable heat block or water bath and a blue light or ultraviolet flashlight for visual detection of MPXV, making it highly accessible. The integration of LAMP and CRISPR/Cas12b provides a robust, user-friendly platform for point-of-care testing, with promising potential for the rapid molecular diagnosis of infectious diseases.},
}

*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Monkeypox virus/isolation & purification/genetics
Humans
*Mpox, Monkeypox/diagnosis/virology
Limit of Detection

@article {pmid40394064,
year = {2025},
author = {Gu, S and Bodai, Z and Anderson, RA and So, HYA and Cowan, QT and Komor, AC},
title = {Elucidating the genetic mechanisms governing cytosine base editing outcomes through CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4685},
pmid = {40394064},
issn = {2041-1723},
support = {T32 GM146648/GM/NIGMS NIH HHS/United States ; T32GM146648//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 27975//Research Corporation for Science Advancement (Research Corporation)/ ; T32GM008326//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MCB-2048207//National Science Foundation (NSF)/ ; T32 GM008326/GM/NIGMS NIH HHS/United States ; },
mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA Repair/genetics ; DNA Mismatch Repair/genetics ; HEK293 Cells ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Cytosine base editors enable programmable and efficient genome editing using an intermediate featuring a U•G mismatch across from a DNA nick. This intermediate facilitates two major outcomes, C•G to T•A and C•G to G•C point mutations, and it is not currently well-understood which DNA repair factors are involved. Here, we couple reporters for cytosine base editing activity with knockdown of 2015 DNA processing genes to identify genes involved in these two outcomes. Our data suggest that mismatch repair factors facilitate C•G to T•A outcomes, while C•G to G•C outcomes are mediated by RFWD3, an E3 ubiquitin ligase. We also propose that XPF, a 3'-flap endonuclease, and LIG3, a DNA ligase, are involved in repairing the intermediate back to the original C•G base pair. Our results demonstrate that competition and collaboration among different DNA repair pathways shape cytosine base editing outcomes.},
}

*Cytosine/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
DNA Repair/genetics
DNA Mismatch Repair/genetics
HEK293 Cells
Ubiquitin-Protein Ligases/metabolism/genetics

@article {pmid40393960,
year = {2025},
author = {Liu, Z and Luan, X and Lu, Q and Qin, S and Zeng, F and Li, Z and He, B and Song, Y},
title = {Reactive oxygen species responsive nanomotors for gene edited metabolic disruption and immunotherapy.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4708},
pmid = {40393960},
issn = {2041-1723},
mesh = {*Reactive Oxygen Species/metabolism ; *Immunotherapy/methods ; *Gene Editing/methods ; Humans ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; Tumor Microenvironment ; Heme Oxygenase-1/metabolism ; *Neoplasms/therapy/immunology/genetics ; CD8-Positive T-Lymphocytes/immunology ; Glycolysis ; Nanoparticles/chemistry ; Female ; },
abstract = {While gene-editing-based tumor therapy holds promise, conventional passive-diffusion vectors face limited penetration in dense solid tumors. Here, we developed a ROS-driven gene editing nanomotor (RDN@PL), which takes hemin as the core and encapsulates CRISPR/Cas9 plasmids targeting LDHA (A glycolysis key enzyme). In tumor microenvironments, RDN@PL consumes extracellular ROS to fuel self-diffusiophoresis, achieving higher intratumoral accumulation than passive particles. Upon internalization, heme oxygenase-1 (HO-1) degrades RDN@PL, releasing CO and plasmids. LDHA knockout suppresses glycolysis while CO elevates mitochondrial ROS, which triggers apoptosis by disrupting metabolism and enhancing immunity. Simultaneously, extracellular ROS depletion by non-internalized nanomotors reverses immunogenic cell death (ICD) inhibition, enhancing CD8+ T cell infiltration in tumor. The Janus nanomotor enables extracellular ROS scavenging and intracellular ROS increment via HO-1-responsive cargo release and gene editing. This multi-level intervention strategy demonstrates 93.9 % tumor growth suppression in solid tumor models, providing a blueprint for engineering intelligent nanovesicles in precision oncology.},
}

*Reactive Oxygen Species/metabolism
*Immunotherapy/methods
*Gene Editing/methods
Humans
Animals
Mice
CRISPR-Cas Systems
Cell Line, Tumor
Tumor Microenvironment
Heme Oxygenase-1/metabolism
*Neoplasms/therapy/immunology/genetics
CD8-Positive T-Lymphocytes/immunology
Glycolysis
Nanoparticles/chemistry
Female

@article {pmid40393243,
year = {2025},
author = {Li, H and Lian, S and Zhang, Z and Bi, W and Meng, Q and Ding, Q},
title = {The CRISPR/Cas13a-assisted electrochemiluminescence sensing device combined with entropy-driven and hybrid chain reaction nucleic acid amplification techniques for ultra-sensitive analysis of brain natriuretic peptide.},
journal = {Talanta},
volume = {295},
number = {},
pages = {128310},
doi = {10.1016/j.talanta.2025.128310},
pmid = {40393243},
issn = {1873-3573},
abstract = {Brain natriuretic peptide (BNP) is considered a reliable marker of heart failure disease, and its timely detection can provide important pathological information to prevent or treat heart failure. In this article, an electrochemiluminescence (ECL) sensing device based on a boron carbon nitride/gold nanoparticle (BCN/AuNPs) complex is developed to determine BNP. Prominently, the CRISPR/CAS 13a enzyme reverse cleavage mode, the entropy-driven and hammer hybridization chain reaction processes were involved in the entire detection scheme. Ultimately, with multiple reaction methods and amplification reactions of nucleic acids, this ECL sensing device is able to achieve a detection limit as low as of 0.03 pg/mL and linear range from 0.1 pg/mL to 30 ng/mL for BNP. In addition, the ECL sensing device based on BCN/AuNPs complex obtained satisfactory stability and specificity, and can also be extended to the detection of other pathological markers.},
}

@article {pmid40389868,
year = {2025},
author = {Amoroso, CG and Andolfo, G},
title = {Hazelnut allergome overview and Cor a gRNAs identification.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {661},
pmid = {40389868},
issn = {1471-2229},
support = {E53D23020840001P//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; },
mesh = {*Corylus/genetics/immunology ; *Allergens/genetics/immunology ; *Plant Proteins/genetics/immunology ; Genome, Plant ; CRISPR-Cas Systems ; Phylogeny ; *Antigens, Plant/genetics/immunology ; },
abstract = {BACKGROUND: Corylus species (hazelnuts) are a valuable source of nutrients and are widely consumed worldwide. Nevertheless, Corylus avellana (Cor a) contains 13 allergens (Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, Cor a 14, Cor a 15, Cor a 16, and Cor a TLP) that have been deposited into the official database (WHO/IUIS) for allergen nomenclature. The recent availability of several Corylus genomes provided opportunities to explore allergome variability, and thus to develop hypoallergenic varieties using modern biotech approaches. Certainly, the identification of CRISPR-Cas9 guide RNA (gRNA) is a pivotal step in achieving this goal. User-friendly web tools include limited reference genomes to design CRISPR-Cas9 gRNAs, while bioinformatic software for custom analysis require advanced command-line skills.

RESULTS: This work explored the evolutionary trajectories of allergenic Cor a homologs in C. avellana, C. americana, C. heterophylla, and C. mandshurica genome assemblies. 52 Cor a orthologs were found in the analyzed species, and a recent tandem duplication of Cor a 1 was found in C. americana. Three new gene models were predicted in C. avellana and C. mandshurica for Cor a 16 and Cor a 10. Additionally, we identified 56 Cor a isoallergens, of which ten Cor a isoforms. Furthermore, phylogenetic analysis sheds light on the evolutionary dynamics of three hazelnut allergens revealing the evolutionary complexity of Cor a 1, Cor a 2, and Cor a TLP within the Corylus genus. A list of multiple gRNAs designed for the CRISPR-Cas9 system was provided for the singular and multiple silencing of Cor a homologs in each Corylus genome.

CONCLUSIONS: This study enhances our knowledge on the evolutionary path of Cor a allergens among Corylus species and provides highly accurate on-target guides targeting hazelnut allergome.},
}

*Corylus/genetics/immunology
*Allergens/genetics/immunology
*Plant Proteins/genetics/immunology
Genome, Plant
CRISPR-Cas Systems
Phylogeny
*Antigens, Plant/genetics/immunology

@article {pmid39893321,
year = {2025},
author = {Gumusgoz, E and Kasiri, S and Youssef, I and Verma, M and Chopra, R and Villarreal Acha, D and Wu, J and Marriam, U and Alao, E and Chen, X and Guisso, DR and Gray, SJ and Shah, BR and Minassian, BA},
title = {Focused ultrasound widely broadens AAV-delivered Cas9 distribution and activity.},
journal = {Gene therapy},
volume = {32},
number = {3},
pages = {237-245},
pmid = {39893321},
issn = {1476-5462},
support = {R01 NS127900/NS/NINDS NIH HHS/United States ; P01NS097197//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Dependovirus/genetics ; Animals ; Mice ; *Genetic Therapy/methods ; Blood-Brain Barrier/metabolism ; Hippocampus/metabolism ; Genetic Vectors/administration & dosage/genetics ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Humans ; Gene Transfer Techniques ; Disease Models, Animal ; },
abstract = {Because children have little temporal exposure to environment and aging, most pediatric neurological diseases are inherent, i.e. genetic. Since postnatal neurons and astrocytes are mostly non-replicating, gene therapy and genome editing present enormous promise in child neurology. Unlike in other organs, which are highly permissive to adeno-associated viruses (AAV), the mature blood-brain barrier (BBB) greatly limits circulating AAV distribution to the brain. Intrathecal administration improves distribution but to no more than 20% of brain cells. Focused ultrasound (FUS) opens the BBB transiently and safely. In the present work we opened the hippocampal BBB and delivered a Cas9 gene via AAV9 intrathecally. This allowed brain first-pass, and subsequent vascular circulation and re-entry through the opened BBB. The mouse model used was of Lafora disease, a neuroinflammatory disease due to accumulations of misshapen overlong-branched glycogen. Cas9 was targeted to the gene of the glycogen branch-elongating enzyme glycogen synthase. We show that FUS dramatically (2000-fold) improved hippocampal Cas9 distribution and greatly reduced the pathogenic glycogen accumulations and hippocampal inflammation. FUS is in regular clinical use for other indications. Our work shows that it has the potential to vastly broaden gene delivery or editing along with clearance of corresponding pathologic basis of brain disease.},
}

*Dependovirus/genetics
Animals
Mice
*Genetic Therapy/methods
Blood-Brain Barrier/metabolism
Hippocampus/metabolism
Genetic Vectors/administration & dosage/genetics
*CRISPR-Associated Protein 9/genetics
Gene Editing/methods
CRISPR-Cas Systems
Humans
Gene Transfer Techniques
Disease Models, Animal

@article {pmid40389577,
year = {2025},
author = {Lv, C and Zhang, F and Ren, L and Zhu, P and Cheng, X and Yang, X and Chen, C and Liu, B},
title = {Rapid visual detection assay for Bactrocera dorsalis (Hendel) using recombinase polymerase amplification and CRISPR/Cas12b.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {17328},
pmid = {40389577},
issn = {2045-2322},
support = {no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; no. 62272060 and 62272061//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Tephritidae/genetics ; *CRISPR-Cas Systems ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {The oriental fruit fly Bactrocera dorsalis (Hendel) is considered as a quarantine pest in many countries and regions. Challenges remain in distinguishing this species with morphological similarities, especially in relevant development stages. In recent years, CRISPR/Cas12b genetic diagnostics has seen rapid advancements and offers an efficient tool for the identification of pathogens, viruses, and other genetic targets. Here we developed a new and rapid visual detection assay of B. dorsalis using recombinase polymerase amplification (RPA) and the CRISPR/Cas12b system. The system can detect different developmental stages of B. dorsalis within 30-35 min at 43 ℃ and the results are easily observed by the naked eye based on the color change in the tube during the reaction. The specificity and high sensitivity of this method was demonstrated, allowing for detection from 3.2 pg µL[- 1] of DNA. With crude DNA, this diagnostic system successfully identified B. dorsalis by holding the reaction tubes in the hand. Our study demonstrates that RPA-CRISPR/Cas12b visualization system is effective to detect B. dorsalis rapidly and accurately. This approach can be applied for monitoring and identification of other pests in border and relevant locations, preventing biological invasions and ensuring pest control.},
}

Animals
*Tephritidae/genetics
*CRISPR-Cas Systems
*Recombinases/metabolism/genetics
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity

@article {pmid40389480,
year = {2025},
author = {Troester, S and Eder, T and Wukowits, N and Piontek, M and Fernández-Pernas, P and Schmoellerl, J and Haladik, B and Manhart, G and Allram, M and Maurer-Granofszky, M and Scheidegger, N and Nebral, K and Superti-Furga, G and Meisel, R and Bornhauser, B and Valent, P and Dworzak, MN and Zuber, J and Boztug, K and Grebien, F},
title = {Transcriptional and epigenetic rewiring by the NUP98::KDM5A fusion oncoprotein directly activates CDK12.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4656},
pmid = {40389480},
issn = {2041-1723},
support = {P35628//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; P35298//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; TAI490//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; },
mesh = {Humans ; *Nuclear Pore Complex Proteins/metabolism/genetics ; *Oncogene Proteins, Fusion/metabolism/genetics ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Cyclin-Dependent Kinases/metabolism/genetics ; *Epigenesis, Genetic ; Cell Line, Tumor ; Animals ; Transcription, Genetic ; Mice ; Gene Expression Regulation, Leukemic ; DNA Damage ; CRISPR-Cas Systems ; Retinoblastoma-Binding Protein 2 ; },
abstract = {Nucleoporin 98 (NUP98) fusion oncoproteins are strong drivers of pediatric acute myeloid leukemia (AML) with poor prognosis. Here we show that NUP98 fusion-expressing AML harbors an epigenetic signature that is characterized by increased accessibility of hematopoietic stem cell genes and enrichment of activating histone marks. We employ an AML model for ligand-induced degradation of the NUP98::KDM5A fusion oncoprotein to identify epigenetic programs and transcriptional targets that are directly regulated by NUP98::KDM5A through CUT&Tag and nascent RNA-seq. Orthogonal genome-wide CRISPR/Cas9 screening identifies 12 direct NUP98::KDM5A target genes, which are essential for AML cell growth. Among these, we validate cyclin-dependent kinase 12 (CDK12) as a druggable vulnerability in NUP98::KDM5A-expressing AML. In line with its role in the transcription of DNA damage repair genes, small-molecule-mediated CDK12 inactivation causes increased DNA damage, leading to AML cell death. Altogether, we show that NUP98::KDM5A directly regulates a core set of essential target genes and reveal CDK12 as an actionable vulnerability in AML with oncogenic NUP98 fusions.},
}

Humans
*Nuclear Pore Complex Proteins/metabolism/genetics
*Oncogene Proteins, Fusion/metabolism/genetics
*Leukemia, Myeloid, Acute/genetics/metabolism/pathology
*Cyclin-Dependent Kinases/metabolism/genetics
*Epigenesis, Genetic
Cell Line, Tumor
Animals
Transcription, Genetic
Mice
Gene Expression Regulation, Leukemic
DNA Damage
CRISPR-Cas Systems
Retinoblastoma-Binding Protein 2

@article {pmid40389438,
year = {2025},
author = {Pandey, AC and Bezney, J and DeAscanis, D and Kirsch, EB and Ahmed, F and Crinklaw, A and Choudhary, KS and Mandala, T and Deason, J and Hamidi, JS and Siddique, A and Ranganathan, S and Brown, K and Armstrong, J and Head, S and Ordoukhanian, P and Steinmetz, LM and Topol, EJ},
title = {A CRISPR/Cas9-based enhancement of high-throughput single-cell transcriptomics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4664},
pmid = {40389438},
issn = {2041-1723},
support = {UL1TR002550//U.S. Department of Health & Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; 20CDA35310187//American Heart Association (American Heart Association, Inc.)/ ; KL2 TR002552/TR/NCATS NIH HHS/United States ; UL1 TR002550/TR/NCATS NIH HHS/United States ; KL2TR002552//U.S. Department of Health & Human Services | NIH | National Center for Advancing Translational Sciences (NCATS)/ ; },
mesh = {*Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptome/genetics ; *Gene Expression Profiling/methods ; Animals ; High-Throughput Nucleotide Sequencing/methods ; Muscle, Smooth, Vascular/cytology/metabolism ; RNA-Seq/methods ; Sequence Analysis, RNA/methods ; Mice ; Myocytes, Smooth Muscle/metabolism ; },
abstract = {Single-cell RNA-seq (scRNAseq) struggles to capture the cellular heterogeneity of transcripts within individual cells due to the prevalence of highly abundant and ubiquitous transcripts, which can obscure the detection of biologically distinct transcripts expressed up to several orders of magnitude lower levels. To address this challenge, here we introduce single-cell CRISPRclean (scCLEAN), a molecular method that globally recomposes scRNAseq libraries, providing a benefit that cannot be recapitulated with deeper sequencing. scCLEAN utilizes the programmability of CRISPR/Cas9 to target and remove less than 1% of the transcriptome while redistributing approximately half of reads, shifting the focus toward less abundant transcripts. We experimentally apply scCLEAN to both heterogeneous immune cells and homogenous vascular smooth muscle cells to demonstrate its ability to uncover biological signatures in different biological contexts. We further emphasize scCLEAN's versatility by applying it to a third-generation sequencing method, single-cell MAS-Seq, to increase transcript-level detection and discovery. Here we show the possible utility of scCLEAN across a wide array of human tissues and cell types, indicating which contexts this technology proves beneficial and those in which its application is not advisable.},
}

*Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
Humans
*Transcriptome/genetics
*Gene Expression Profiling/methods
Animals
High-Throughput Nucleotide Sequencing/methods
Muscle, Smooth, Vascular/cytology/metabolism
RNA-Seq/methods
Sequence Analysis, RNA/methods
Mice
Myocytes, Smooth Muscle/metabolism

@article {pmid40389165,
year = {2025},
author = {Öktem, M and Nguyen, TH and Bosman, EDC and Fens, MHAM and Caiazzo, M and Mastrobattista, E and Lei, Z and de Jong, OG},
title = {Lipopeptide-mediated delivery of CRISPR/Cas9 ribonucleoprotein complexes for gene editing and correction.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {383},
number = {},
pages = {113854},
doi = {10.1016/j.jconrel.2025.113854},
pmid = {40389165},
issn = {1873-4995},
abstract = {CRISPR/Cas gene editing is a highly promising technology for the treatment and even potential cure of genetic diseases. One of the major challenges for its therapeutic use is finding safe and effective vehicles for intracellular delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex. In this study, we tested and characterized a series of novel fatty acid-modified versions of a previously reported Cas9 RNP carrier, consisting of a complex of the cell-penetrating peptide (CPP) LAH5 with Cas9 RNP and homology-directed DNA repair templates. Comparative experiments demonstrated that RNP/peptide nanocomplexes showed various improvements depending on the type of fatty acid modification. These improvements included enhanced stability in serum, improved membrane disruption capability and increased transfection efficacy. Cas9 RNP/oleic acid LAH5 peptide nanocomplexes showed the overall best performance for both gene editing and correction. Moreover, Cas9 RNP/oleic acid LAH5 nanocomplexes significantly protected the Cas9 protein cargo from enzymatic protease digestion. In addition, in vivo testing demonstrated successful gene editing after intramuscular administration. Despite the inherent barriers of the tightly organized muscle tissues, we achieved approximately 10 % gene editing in the skeletal muscle tissues when targeting the CAG-tdTomato locus in the transgenic Ai9 Cre-LoxP reporter mouse strain and 7 % gene editing when targeting the Ccr5 gene, without any observable short-term toxicity. In conclusion, the oleic acid-modified LAH5 peptide is an effective delivery platform for direct Cas9/RNP delivery, and holds great potential for the development of new CRISPR/Cas9-based therapeutic applications for the treatment of genetic diseases.},
}

@article {pmid40388365,
year = {2025},
author = {Wang, Y and Hu, J and Zhang, J},
title = {Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {219},
pages = {},
doi = {10.3791/67508},
pmid = {40388365},
issn = {1940-087X},
mesh = {Animals ; *Hematopoietic Stem Cells/cytology/physiology/metabolism ; Mice ; *CRISPR-Cas Systems ; Tumor Suppressor Protein p53/genetics ; Cell Proliferation ; Transfection ; },
abstract = {Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.},
}

Animals
*Hematopoietic Stem Cells/cytology/physiology/metabolism
Mice
*CRISPR-Cas Systems
Tumor Suppressor Protein p53/genetics
Cell Proliferation
Transfection

@article {pmid40359808,
year = {2025},
author = {Liu, S and Xiao, G and Li, P and Xu, Y and Fan, X and Tian, L and Zhang, S and Zhang, G},
title = {Plasma-based ultrasensitive detection of Mycobacterium tuberculosis ESAT6/CFP10 fusion antigen using a CRISPR-driven aptamer fluorescence testing (CRAFT).},
journal = {Biosensors & bioelectronics},
volume = {284},
number = {},
pages = {117566},
doi = {10.1016/j.bios.2025.117566},
pmid = {40359808},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/isolation & purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Bacterial Proteins/blood/genetics/isolation & purification ; *Aptamers, Nucleotide/chemistry/genetics ; *Antigens, Bacterial/blood/genetics/isolation & purification ; *Tuberculosis/diagnosis/microbiology/blood ; Limit of Detection ; CRISPR-Cas Systems ; Fluorescence ; SELEX Aptamer Technique ; },
abstract = {Tuberculosis (TB) screening in clinical diagnosis is challenging due to issues such as sputum dependence, time-consuming procedures, and high costs. In this study, we introduce a CRAFT (CRISPR-Driven Aptamer Fluorescence Testing), an aptamer-based CRISPR/Cas12a assay designed for the rapid and sensitive detection of Mycobacterium tuberculosis (Mtb) antigens from peripheral blood. Aptamer 3 (Ap3) and the aptamer-mediated probe (Aptamer-blocker 3-7) were selected through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Ap3 demonstrated a dissociation constant (Kd) of 8.3E-7 M with the ESAT6/CFP10 fusion proteins (EC proteins), which are produced during the replicative phase of Mtb. Upon labeling the EC proteins with Aptamer-blocker 3-7 (Ap-blocker 3-7) probe, single-stranded DNA (ssDNA) blocker 3-7 was released, thereby completing the process for RPA-based CRISPR/Cas12a fluorescence detection. After optimizing multiple parameters, CRAFT achieved a detection limit of 0.1 ag/mL EC proteins (equivalent to 3 protein particles per mL) within 120 min from plasma sample to result. The method was validated with 86 clinical plasma samples confirmed the method's high diagnostic accuracy for Mtb infection (sensitivity: 97.1 %, 95 % confidence interval (CI) [0.849-0.998]); specificity: 98.0 %, 95 % CI [0.897-0.999]), supporting its utility in early therapeutic evaluation of tuberculosis management.},
}

*Mycobacterium tuberculosis/isolation & purification/genetics
Humans
*Biosensing Techniques/methods
*Bacterial Proteins/blood/genetics/isolation & purification
*Aptamers, Nucleotide/chemistry/genetics
*Antigens, Bacterial/blood/genetics/isolation & purification
*Tuberculosis/diagnosis/microbiology/blood
Limit of Detection
CRISPR-Cas Systems
Fluorescence
SELEX Aptamer Technique

@article {pmid40306523,
year = {2025},
author = {Sui, B and Liu, C and Sun, Z and Zheng, Y and Zhou, W and Liu, H},
title = {CRISPR/Cas12a-based transition state molecular switch for low-background detection of HPV-16 on a microfluidic platform.},
journal = {International journal of biological macromolecules},
volume = {311},
number = {Pt 1},
pages = {143556},
doi = {10.1016/j.ijbiomac.2025.143556},
pmid = {40306523},
issn = {1879-0003},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; Biosensing Techniques/methods ; *Lab-On-A-Chip Devices ; *Microfluidics/methods ; DNA, Viral/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human papillomavirus type 16 (HPV-16) is a high-risk oncogenic subtype of HPV, strongly associated with the pathogenesis of multiple cancers. Researchers have developed many detection methods for HPV-16, among which, the detection method based on microfluidic has the characteristics of high efficiency and high sensitivity. However, non-specific adsorption remains a critical challenge, often leading to elevated background signals. Here, we propose an On-Chip assay integrated by transition state molecular switch based on CRISPR-Cas12a (OCTMS-CRISPR) for stable, sensitive, and low-background fluorescence detection of HPV-16. This system leverages a highly integrated molecular switch and Cas12a to perform dual-screening, while the dissociation products of the molecular switch activate trans-cleavage activity. Our data suggest that OCTMS-CRISPR suppresses background signals on microfluidic chip while maintaining the specificity and sensitivity of trans-cleavage. For demonstration, we detected five HPV subtypes and base mismatches at varying positions and quantities. The LOD can reach 7.64 pM (average fluorescence intensity) and 9.91fM (pixel counting), showing great potential in the field of biosensing and DNA chips.},
}

*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
Biosensing Techniques/methods
*Lab-On-A-Chip Devices
*Microfluidics/methods
DNA, Viral/genetics
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40300299,
year = {2025},
author = {Lu, L and Gan, S and Xiao, S and Mu, X and Zhao, S and Tian, J},
title = {An innovative "double-locked" CRISPR/Cas12a system based on DNAzyme for the precise imaging of microRNAs in living cells.},
journal = {International journal of biological macromolecules},
volume = {310},
number = {Pt 4},
pages = {143605},
doi = {10.1016/j.ijbiomac.2025.143605},
pmid = {40300299},
issn = {1879-0003},
mesh = {*MicroRNAs/genetics/metabolism ; *DNA, Catalytic/metabolism/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Optical Imaging/methods ; *Molecular Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {We constructed a "double-locked" CRISPR/Cas12a system based on DNAzyme for miRNA detection and precise imaging. One lock function to close the catalytic activity of DNAzyme, while the other lock serves to inhibit the cleavage function of CRISPR-induced RNA (crRNA). This "double-lock" mechanism ensures that the system is inhibited in the absence of the target molecule miRNA-141, effectively reducing the background signal. When the target miRNA-141 is present, the "lock" of DNAzyme is opened, and DNAzyme further opens the "lock" of crRNA, which activates the trans-cleavage ability of Cas12a on F-Q probe, and the fluorescence signal is restored. The linear range of miRNA-141 was 50 pmol/L ~ 15 nmol/L, and the detection limit was 47 pmol/L (S/N = 3). The system has been successfully applied to detect miRNA-141 expression levels in cell lysates. Meanwhile, this method can be applied for intracellular miRNA-141 fluorescence imaging and fluctuations in intracellular miRNA-141 expression. Overall, this strategy not only offers new prospects for programmable Cas12a detection systems, but also provides new insights for early diagnosis and screening of diseases by combining this in vitro assay with live cell imaging analysis for the detection of cancer markers.},
}

*MicroRNAs/genetics/metabolism
*DNA, Catalytic/metabolism/genetics
Humans
*CRISPR-Cas Systems/genetics
Optical Imaging/methods
*Molecular Imaging/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40286970,
year = {2025},
author = {Long, K and Liu, H and Yang, N and Li, J and Hou, C and Yang, M and Huang, Z and Huo, D},
title = {Selenium-enhanced recombinase polymerase amplification-CRISPR/Cas12a: A low-noise single-tube assay for human papillomavirus 16 ultrasensitive detection.},
journal = {International journal of biological macromolecules},
volume = {310},
number = {Pt 4},
pages = {143468},
doi = {10.1016/j.ijbiomac.2025.143468},
pmid = {40286970},
issn = {1879-0003},
mesh = {*Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Selenium/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; DNA, Viral/genetics ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; },
abstract = {In vitro amplification represents a critical step in human papillomavirus (HPV) DNA detection. However, DNA polymerases can initiate nonspecific amplification and incorporate erroneous nucleotides due to the lack of cellular repair mechanisms. To address these challenges, we present a novel one-tube selenium-enhanced recombinase polymerase amplification (Se-RPA) coupled with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 12a (Cas12a) (OTSRC) system for the ultrasensitive HPV DNA detection. The Se-RPA incorporates 10 % selenium-modified nucleoside triphosphates (dNTPαSe) into the conventional RPA protocol, effectively suppressing nonspecific amplification while maintaining high-fidelity DNA synthesis. The CRISPR/Cas12a component integrates sequence-specific verification, exponential signal amplification, and fluorescence-based readout capabilities. Optimized in a single-tube format to minimize aerosol contamination, OTSRC exhibits a background signal of 71.77 % compared to the one-tube RPA-CRISPR/Cas12a (OTRC) system. Within a 20-min incubation, the OTSRC demonstrated a detection limit of 169 aM, which is half that of the OTRC without dNTPαSe and comparable to qPCR. Furthermore, the OTSRC system demonstrates the excellent compatibility of dNTPαSe with the RPA-CRISPR/Cas12a system, thereby enhancing HPV detection sensitivity. Overall, OTSRC enables rapid, sensitive, and specific detection of HPV DNA, showing strong potential for clinical point-of-care nucleic acid testing applications.},
}

*Human papillomavirus 16/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Selenium/chemistry
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
DNA, Viral/genetics
Limit of Detection
*Endodeoxyribonucleases/genetics/metabolism
*Papillomavirus Infections/diagnosis/virology
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins

@article {pmid40267574,
year = {2025},
author = {Zhang, D and Gao, T and Guo, Q and Ren, Y and Zhu, W and Zhuang, H and Pan, Z and Wang, F and Chen, Y and Guo, M and Liu, T and Wang, C and Ji, L and Qian, W and Li, J and Hou, S and Sun, Z and Wang, X and Xu, J and Guo, H},
title = {Rapid and sensitive Mycoplasma detection in antibody bioprocessing via RPA-CRISPR/Cas12a.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {263},
number = {},
pages = {116904},
doi = {10.1016/j.jpba.2025.116904},
pmid = {40267574},
issn = {1873-264X},
mesh = {*Mycoplasma/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Antibodies, Monoclonal ; *Nucleic Acid Amplification Techniques/methods ; RNA, Ribosomal, 16S/genetics ; Drug Contamination/prevention & control ; Recombinases/metabolism ; Sensitivity and Specificity ; },
abstract = {Mycoplasma species are prevalent microbial contaminants in the production of biological products, such as monoclonal antibodies, posing significant threats to the safety and efficacy of these products. Current regulatory guidelines as well as pharmacopoeias mandate the demonstration of the absence of Mycoplasma in the cell culture and further downstream processing to ensure product safety. Despite recent advancements in sensitive detection techniques for Mycoplasma in eucaryotic expression systems, these methods remain complex and time-consuming. There is a pressing need for a rapid, simple, and sensitive process analytical technology (PAT) for Mycoplasma detection. Here, we report the first development and application of a recombinase polymerase amplification (RPA)-assisted CRISPR-Cas12a (RPA-CRISPR/Cas12a) system spcifically tailored for Mycoplasma detection in biopharmaceutical production. This system combines the high-sensitivity isothermal nucleic acid amplification capabilities of RPA with the trans-cleavage activity of CRISPR-Cas12a reporter probes, enabling the rapid and accurate detection of Mycoplasma, accommodating various experimental requirements and application scenarios. By designing RPA universal primers and crRNA targeting the highly conserved sequences of Mycoplasma 16S rRNA and optimizing reaction conditions, we achieved dual-specific recognition with unprecedented efficiency in bioprocessing samples. All tested Mycoplasma specimens were detectable with limits between 10 and 0.1 copies/μL, with the whole process taking less than 1 hour. We further evaluated the feasibility of this method in detecting Mycoplasma in the cell culture of antibody products and further downstream processing samples. This method reduces the risk of false-positive signals due to non-specific amplification, enhancing detection sensitivity and specificity while significantly reducing analysis, representing the first PAT-compatible method for rapid Mycoplasma monitoring in antibody manufacturing, thereby providing robust assurance for the quality and safety of biological products.},
}

*Mycoplasma/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Antibodies, Monoclonal
*Nucleic Acid Amplification Techniques/methods
RNA, Ribosomal, 16S/genetics
Drug Contamination/prevention & control
Recombinases/metabolism
Sensitivity and Specificity

@article {pmid40387535,
year = {2025},
author = {Liu, S and Yu, T and Song, L and Kalantar-Zadeh, K and Liu, G},
title = {CRISPR/Cas on Microfluidic Paper-Based Analytical Devices for Point-of-Care Screening of Cervical Cancer.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00863},
pmid = {40387535},
issn = {2379-3694},
abstract = {Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.},
}

@article {pmid40183844,
year = {2025},
author = {Lai, S and Guo, J and Li, X and Yu, X and Lai, G},
title = {Dual DNA recycling amplification-assisted CRISPR/Cas12a cleavage for dual-channel ratiometric fluorescence biosensing of kanamycin antibiotic.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {14},
pages = {3191-3200},
pmid = {40183844},
issn = {1618-2650},
support = {22076043//National Natural Science Foundation of China/ ; 2023AFD003//Natural Science Foundation of Hubei Province/ ; },
mesh = {*Kanamycin/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; *DNA/chemistry/genetics ; Milk/chemistry ; Fluorescence ; *CRISPR-Associated Proteins/metabolism ; Spectrometry, Fluorescence/methods ; Nucleic Acid Amplification Techniques/methods ; Aptamers, Nucleotide/chemistry ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Fluorescence biosensors hold significant importance for testing antibiotic residues which seriously endanger public health. However, how to adopt appropriate strategies to address the false result disadvantage involved in traditional single-channel biosensors is still a great challenge. Meanwhile, too much attention focused on designing signal amplification strategies of biosensors unavoidably decreases their detection efficiency. Herein, we combined the designed dual DNA recycling amplification strategy with CRISPR/Cas12a-mediated dual-channel signal output mode to successfully develop a novel ratiometric fluorescence biosensor for testing kanamycin (Kana) residues in complex sample matrices. The first recycling was formed from an exonuclease-assisted aptamer recognition reaction, which also triggered another cascade DNA recycling to amplify the release of the Cas12a activator. With the non-discrimination cleavage of Cas12a to cause reverse fluorescence changes of copper nanoclusters and an AMAC-labeled signal DNA, the ratiometric signal transduction strategy was constructed. Under optimal conditions, this biosensor could be applied for ultrasensitive testing of Kana antibiotics in a five-order of magnitude wide linear range with a low detection limit of 17.2 fg mL[-1]. Benefiting from the self-correction function of the ratiometric signal transduction mode, it showed promising practicality in lake water and milk samples with the relative error less than 4.9% to the standard ELISA results. Besides CRISPR/Cas12a-based fluorescence output efficiency improvement, this biosensor also excluded the complicated manipulations and expensive instruments required in traditional methods. Therefore, it provides a good choice for expanding the application of fluorescence biosensing technology for practical analysis application.},
}

*Kanamycin/analysis
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Anti-Bacterial Agents/analysis
Limit of Detection
*DNA/chemistry/genetics
Milk/chemistry
Fluorescence
*CRISPR-Associated Proteins/metabolism
Spectrometry, Fluorescence/methods
Nucleic Acid Amplification Techniques/methods
Aptamers, Nucleotide/chemistry
Animals
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40387367,
year = {2025},
author = {Li, J and Liu, Y and Jiang, J and Chen, F and Zhang, N and Kang, X and Liu, L and Wang, Y and Xia, Q and Zhu, C and Kuang, D},
title = {Type I-E* CRISPR-Cas of Klebsiella pneumoniae upregulates bacterial virulence by targeting endogenous histidine utilization system.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0021525},
doi = {10.1128/msphere.00215-25},
pmid = {40387367},
issn = {2379-5042},
abstract = {Klebsiella pneumoniae is a globally recognized microbial pathogen with significant clinical impact. The bacterium harbors the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems, which provide adaptive immunity against invading foreign nucleic acids. Recent studies suggest that certain CRISPR-Cas systems can regulate endogenous genes, influencing bacterial virulence. However, their role in regulating pathogenicity in K. pneumoniae remains poorly understood. This study investigates the regulatory role of the type I-E* CRISPR-Cas system in a hypervirulent K. pneumoniae strain, focusing on its impact on histidine metabolism and pathogenicity. Transcriptome analyses identified differentially expressed genes (DEGs) between the casABECD-deletion and wild-type strains, including significant upregulation of the histidine utilization (Hut) operon and downregulation of biofilm-related genes. These molecular changes resulted in enhanced histidine metabolic activity, reduced biofilm formation, attenuated virulence in A549 lung epithelial cells, and improved survival of Galleria mellonella, as validated through phenotypic and virulence assays. Our bioinformatic analysis indicated that the CRISPR-Cas system in K. pneumoniae targets the hutT sequence, which is part of the Hut operon. Furthermore, the overexpression of hutT mitigated CRISPR-Cas-mediated repression of the Hut operon, as observed in virulence assays, while simultaneous deletion of hutH and casABECD restored the reduced virulence in the ΔcasABECD strain. Additionally, deletion of casABECD significantly enhances the growth of the strain in medium with histidine as the sole carbon source, highlighting the intricate regulatory role of the CRISPR-Cas system in metabolic adaptation. Collectively, these findings uncover a novel role for the CRISPR-Cas system in regulating metabolic pathways and virulence in hypervirulent K. pneumoniae.IMPORTANCEClustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are primarily recognized for their roles in adaptive immunity against foreign genetic elements in bacteria. However, emerging evidence indicates that these systems can also regulate endogenous genes, thereby influencing bacterial physiology and virulence. In this study, we demonstrate that the type I-E* CRISPR-Cas system in Klebsiella pneumoniae targets the hutT gene, a critical component of the histidine utilization (Hut) pathway. This targeting potentially impacts hutT transcription and alters the expression of other hut genes, ultimately enhancing bacterial virulence. Our findings reveal a previously unrecognized regulatory mechanism through which CRISPR-Cas systems facilitate metabolic adaptation and pathogenicity in K. pneumoniae. This study broadens our understanding of the multifaceted roles of CRISPR-Cas systems in bacterial physiology and pathobiology, with implications for clinically relevant pathogens.},
}

@article {pmid40387084,
year = {2025},
author = {Belato, HB and Knight, AL and D'Ordine, AM and Pindi, C and Fan, Z and Luo, J and Palermo, G and Jogl, G and Lisi, GP},
title = {Structural and dynamic impacts of single-atom disruptions to guide RNA interactions within the recognition lobe of Geobacillus stearothermophilus Cas9.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40387084},
issn = {2050-084X},
support = {P30 GM133893/GM/NIGMS NIH HHS/United States ; R01GM141329/NH/NIH HHS/United States ; R01GM136815/NH/NIH HHS/United States ; R01 GM136815/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; CHE2144823//National Science Foundation/ ; R01 GM141329/GM/NIGMS NIH HHS/United States ; MCB2143760//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; },
mesh = {*Geobacillus stearothermophilus/enzymology/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Protein Conformation ; },
abstract = {The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.},
}

*Geobacillus stearothermophilus/enzymology/genetics
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
*CRISPR-Associated Protein 9/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/chemistry/genetics
CRISPR-Cas Systems
Protein Conformation

@article {pmid40380978,
year = {2025},
author = {Pipes, BL and Nishiguchi, MK},
title = {Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis.},
journal = {Archives of microbiology},
volume = {207},
number = {7},
pages = {147},
pmid = {40380978},
issn = {1432-072X},
support = {EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; EXO 80NSSC18K1053//National Aeronautical and Space Adminstration/ ; DBI-2214028//National Science Foundation/ ; DBI-2214028//National Science Foundation/ ; },
mesh = {*Aliivibrio fischeri/genetics/physiology ; *Symbiosis/genetics ; Animals ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; *Decapodiformes/microbiology ; Genetic Vectors ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The Vibrio fischeri-Euprymna scolopes symbiosis has become a powerful animal-microbe model system to examine the genetic underpinnings of symbiont development and regulation. Although there has been a number of elegant bacterial genetic technologies developed to examine this symbiosis, there is still a need to develop more sophisticated methodologies to better understand complex regulatory pathways that lie within the association. Therefore, we have developed a suite of CRISPR interference (CRISPRi) vectors for inducible repression of specific V. fischeri genes associated with symbiotic competence. The suite utilizes both Tn7-integrating and shuttle vector plasmids that allow for inducible expression of CRISPRi dCas9 protein along with single-guide RNAs (sgRNA) modules. We validated this CRISPRi tool suite by targeting both exogenous (an introduced mRFP reporter) and endogenous genes (luxC in the bioluminescence producing lux operon, and flrA, the major regulatory gene controlling flagella production). The suite includes shuttle vectors expressing both single and multiple sgRNAs complementary to the non-template strand of multiple targeted genetic loci, which were effective in inducible gene repression, with significant reductions in targeted gene expression levels. V. fischeri cells harboring a version of this system targeting the luxC gene and suppressing the production of luminescence were used to experimentally validate the hypothesis that continuous luminescence must be produced by the symbiont in order to maintain the symbiosis at time points longer than the known 24-h limit. This robust new CRISPRi genetic toolset has broad utility and will enhance the study of V. fischeri genes, bypassing the need for gene disruptions by standard techniques of allelic knockout-complementation-exchange and the ability to visualize symbiotic regulation in vivo.},
}

*Aliivibrio fischeri/genetics/physiology
*Symbiosis/genetics
Animals
*Gene Expression Regulation, Bacterial
*CRISPR-Cas Systems
*Decapodiformes/microbiology
Genetic Vectors
Bacterial Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Plasmids/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40380066,
year = {2025},
author = {Garduño-Tamayo, YQ and Acosta-García, G},
title = {Gene Silencing in Plants Through Exogenous Application of miRNAs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {249-255},
pmid = {40380066},
issn = {1940-6029},
mesh = {*MicroRNAs/genetics ; *Gene Silencing ; *Plants/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; RNA Interference ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; RNA, Plant/genetics ; },
abstract = {Although omics technologies allow us to identify genes involved in various biological processes, we still rely on the analysis of the function of individual genes. Studies of the function of a specific gene include technologies such as gene silencing by RNAi or genome editing by CRISPR-cas9. However, most of them depend on the availability of transformation methods, so they are not established for all plants. In this chapter, we report a protocol that involves the exogenous application of miRNAs for the specific silencing of genes of interest. The advantage of this protocol is that it does not require a transformation event and can be applied to a certain tissue or developmental stage. This novel technology facilitates the analysis of specific gene functions in crops of economic interest.},
}

*MicroRNAs/genetics
*Gene Silencing
*Plants/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems
RNA Interference
Gene Editing/methods
Plants, Genetically Modified/genetics
RNA, Plant/genetics

@article {pmid40380065,
year = {2025},
author = {Rivera-Toro, DM and Alvarez-Venegas, R},
title = {CRISPR-Activation: Boosting Expression of Plant MIRs.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {229-247},
pmid = {40380065},
issn = {1940-6029},
mesh = {*MicroRNAs/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Plant ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Plants/genetics ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.},
}

*MicroRNAs/genetics
*CRISPR-Cas Systems
*Gene Expression Regulation, Plant
*Gene Editing/methods
Promoter Regions, Genetic
*Plants/genetics
Plants, Genetically Modified/genetics
Genetic Vectors/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40380064,
year = {2025},
author = {Martínez-Estrada, E and de la Mora-Franco, D and de Folter, S},
title = {A Simple and Efficient Protocol to Transform and Regenerate CRISPR-Cas9-Mediated Genome-Edited Tomato Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2900},
number = {},
pages = {213-228},
pmid = {40380064},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Cotyledon/genetics/growth & development ; Transformation, Genetic ; Agrobacterium/genetics ; },
abstract = {CRISPR-Cas9-mediated genome editing has revolutionized functional genomics and crop improvement. However, to maximize the adoption of the CRISPR-Cas9 technology, an efficient method to transform and regenerate genetically edited plants is necessary. In this protocol, we describe a detailed method to generate a CRISPR-Cas9 construct based on the Golden Gate cloning system and a simple and efficient method to transform and regenerate tomato plants from cotyledons co-cultured with Agrobacterium. Our protocol allows the production of at least ten Cas-positive independent lines from one hundred cotyledons. This protocol is routinely used in our laboratory to obtain tomato mutant lines and has been proven effective across several genotypes.},
}

*Solanum lycopersicum/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genome, Plant
Plants, Genetically Modified/genetics
Cotyledon/genetics/growth & development
Transformation, Genetic
Agrobacterium/genetics

@article {pmid40312015,
year = {2025},
author = {Li, H and Li, C and Pedro, GC and Zhang, Q and Jiang, H and Yang, Y and Duan, Y and Sun, X},
title = {CRISPR/Cas9-mediated gene editing of MsCLE3a confers compact alfalfa architecture.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {357},
number = {},
pages = {112532},
doi = {10.1016/j.plantsci.2025.112532},
pmid = {40312015},
issn = {1873-2259},
mesh = {*Medicago sativa/genetics/growth & development/anatomy & histology ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; Plant Leaves/genetics/growth & development/anatomy & histology ; Gene Expression Regulation, Plant ; Brassinosteroids/metabolism ; },
abstract = {Alfalfa (Medicago sativa L.) is a globally major forage crop for livestock due to its superior nutritional value and palatability. As a premium forage species, alfalfa breeding has traditionally emphasized high yield. In this study, we designed guide RNA (gRNA) to target the coding sequence of the MsCLE3a gene, and constructed a CRISPR/Cas9 vector to generate knockouts. Five mutant lines were successfully isolated, displaying upright petioles and a compact ternately compound leaf phenotype during the seedling stage. Cryo-scanning electron microscopy (Cryo-SEM) was employed to analyze cell morphology on the abaxial leaf surface. The results indicated that the cells on the abaxial side of the Mscle3a mutants were significantly elongated compared to the wild type (WT). Transcriptomic analysis further uncovered the down-regulation of brassinosteroid (BR) biosynthesis genes in Mscle3a mutants, which might underlie the mechanisms regulating petiole angle. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a major reduction in the transcript levels of several key genes involved in BR signaling pathways in the Mscle3a mutants relative to WT. This transcriptional down-regulation was strongly associated with the observed alterations in leaf petiole angle (LPA) in the mutant lines. Our findings provide novel insights into the novel regulatory role of MsCLE3a in alfalfa development and pinpoint potential targets for manipulating LPA in crop breeding programs.},
}

*Medicago sativa/genetics/growth & development/anatomy & histology
*CRISPR-Cas Systems/genetics
*Gene Editing
*Plant Proteins/genetics/metabolism
Plant Leaves/genetics/growth & development/anatomy & histology
Gene Expression Regulation, Plant
Brassinosteroids/metabolism

@article {pmid40269535,
year = {2025},
author = {Abe, T and Inoue, KI and Kiyonari, H},
title = {Efficient CRISPR/Cas9-mediated knockin of reporter genes in rats at ROSA26 by pronuclear microinjection.},
journal = {Development, growth & differentiation},
volume = {67},
number = {4},
pages = {215-225},
doi = {10.1111/dgd.70007},
pmid = {40269535},
issn = {1440-169X},
support = {//RIKEN Intramural funding/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Rats ; Microinjections/methods ; *Genes, Reporter/genetics ; *Gene Knock-In Techniques/methods ; Female ; Zygote/metabolism ; Gene Editing/methods ; Rats, Transgenic ; },
abstract = {The genetic modification of rats is a key technology for advancing biomedical research on human diseases. CRISPR/Cas9-mediated genome editing enables the generation of knockout rats in a single step, without the need for embryonic stem cells, by directly injecting genome editing components into zygotes. This simplifies the process, reduces costs, and accelerates gene function analysis in rats. However, the insertion of a gene cassette into a target site has remained inefficient, limiting the generation of knockin (KI) rats. To overcome this issue, we developed an optimized method that covers the entire process from zygote harvesting with superovulation to timed microinjection, ensuring the consistent generation of KI rats. We successfully generated four different fluorescent reporter lines at the ROSA26 locus in rats. Our study provides detailed, step-by-step protocols for donor vector design, zygote collection, microinjection, founder screening, and cryopreservation in rats.},
}

Animals
*CRISPR-Cas Systems/genetics
Rats
Microinjections/methods
*Genes, Reporter/genetics
*Gene Knock-In Techniques/methods
Female
Zygote/metabolism
Gene Editing/methods
Rats, Transgenic

@article {pmid40207549,
year = {2025},
author = {Bu, Y and Wang, R and Liu, Y and Xing, K and Zhang, X and Sun, Y and Zhang, J},
title = {CRISPR/Cas9-mediated myostatin disruption elevates the expression of genes associated with myofiber composition and growth in Exopalaemon carinicauda.},
journal = {The Journal of experimental biology},
volume = {228},
number = {10},
pages = {},
doi = {10.1242/jeb.250148},
pmid = {40207549},
issn = {1477-9145},
support = {32373121//Innovative Research Group Project of the National Natural Science Foundation of China/ ; 22323201D//Hebei Provincial Key Research Projects/ ; HBU2023BS027//Hebei Province Graduate Innovation Funding Project/ ; D2023201002//Steel and Iron Foundation of Hebei Province/ ; 32373121//National Natural Science Foundation of China/ ; 32172954//National Natural Science Foundation of China/ ; 22323201D//Key Research and Development Project of Hebei Province/ ; HBU2023BS027//Post-graduate Innovation Fund Project of Hebei University/ ; D2023201002//Natural Science Foundation of Hebei Province/ ; },
mesh = {Animals ; *Myostatin/genetics/metabolism ; *CRISPR-Cas Systems ; *Palaemonidae/genetics/growth & development/metabolism ; *Arthropod Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Myostatin (MSTN) is a negative regulator of skeletal muscle development and growth in vertebrates, but its role in crustaceans remains debated. To explore the functional role of MSTN in Exopalaemon carinicauda (EcMSTN) and to facilitate the development of new strains with enhanced growth rates, we investigated the molecular characteristics, expression patterns and functional implications of EcMSTN. We employed CRISPR/Cas9-mediated gene editing technology to generate EcMSTN knockout (EcMSTN-KO) prawns and subsequently monitored their hatching rate, survival rate and growth performance. The findings revealed that the hatching rate in the EcMSTN-KO group was only 11%, significantly lower than the 50% in the control group (P<0.05). In comparison to their wild-type (WT) siblings (1.212±0.114 cm), the EcMSTN-KO prawns (1.481±0.192) demonstrated a markedly enhanced body length (P<0.001). The expression of genes associated with myofiber composition and growth, including myosin heavy chain 2 (EcMHC2) and myosin light chain 1 (EcMLC1), exhibited a highly significant increase (P<0.001) in EcMSTN-KO prawns. Additionally, the expression of ecdysone receptor (EcEcR), a molt-related gene, was significantly elevated (P<0.001), while the expression of retinoid X receptor (EcRXR) showed no significant difference (P>0.05). The above studies indicate that EcMSTN functions as a negative regulator of muscle growth in E. carinicauda. Moreover, EcMSTN may play a role in molting. These results underscore the significant potential of MSTN as a genetic target for improving crustacean aquaculture, particularly through gene editing technologies aimed at enhancing growth traits.},
}

Animals
*Myostatin/genetics/metabolism
*CRISPR-Cas Systems
*Palaemonidae/genetics/growth & development/metabolism
*Arthropod Proteins/genetics/metabolism
Gene Editing

@article {pmid40177811,
year = {2025},
author = {Emran, F and Kays, I and Lo, CA and Li, Y and Chen, BE},
title = {A drug screening platform for protein expression levels in neurological disorders.},
journal = {BioTechniques},
volume = {77},
number = {3},
pages = {113-124},
doi = {10.1080/07366205.2025.2484094},
pmid = {40177811},
issn = {1940-9818},
mesh = {Humans ; *Nervous System Diseases/metabolism/genetics/drug therapy ; Drug Evaluation, Preclinical/methods ; *High-Throughput Screening Assays/methods ; CRISPR-Cas Systems ; Gene Editing ; HEK293 Cells ; *Proteins/genetics/metabolism/analysis ; },
abstract = {Neurological and psychiatric diseases and disorders affect more than half of the population. Many of these diseases are caused by the malfunctioning of protein synthesis, where too little or too much production of a protein harms a cell and its functions within the brain. We developed a drug screening platform to identify compounds that target the primary cause of these diseases, namely protein expression amounts. This cellular assay monitors protein expression of a target disease gene along with the protein expression of a control gene using the Protein Quantitation Ratioing (PQR) technique. PQR tracks protein concentration using fluorescence. We used human cells and CRISPR-Cas9 genome editing to insert the Protein Quantitation Reporter into target genes. These cells are used in high-throughput drug screening measuring the fluorescence as the assay. Drug hits can be validated using the same PQR technique or animal models of the disease.},
}

Humans
*Nervous System Diseases/metabolism/genetics/drug therapy
Drug Evaluation, Preclinical/methods
*High-Throughput Screening Assays/methods
CRISPR-Cas Systems
Gene Editing
HEK293 Cells
*Proteins/genetics/metabolism/analysis

@article {pmid40379664,
year = {2025},
author = {Nam, H and Xie, K and Majumdar, I and Wang, J and Yang, S and Starzyk, J and Lee, D and Shan, R and Li, J and Wu, H},
title = {Engineering tripartite gene editing machinery for highly efficient non-viral targeted genome integration.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4569},
pmid = {40379664},
issn = {2041-1723},
support = {DP2 GM154019/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Transgenes/genetics ; DNA, Single-Stranded/genetics/metabolism ; HEK293 Cells ; Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; DNA, Circular/genetics ; Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Homologous Recombination ; Gene Knock-In Techniques/methods ; },
abstract = {Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
Transgenes/genetics
DNA, Single-Stranded/genetics/metabolism
HEK293 Cells
Receptors, Chimeric Antigen/genetics/metabolism
T-Lymphocytes/metabolism
DNA, Circular/genetics
Genetic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics
Homologous Recombination
Gene Knock-In Techniques/methods

@article {pmid40378977,
year = {2025},
author = {Qin, B and Shen, S and Chen, H and Wang, Y and Ding, J and Ding, J},
title = {Inactivation of the key ORFs of HBV for antiviral therapy by non-cleavage base editing.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107689},
doi = {10.1016/j.micpath.2025.107689},
pmid = {40378977},
issn = {1096-1208},
abstract = {OBJECTIVES: Hepatitis B virus (HBV) infection is the key cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Currently available anti-HBV drugs are more or less defective owing to the unremovable covalently closed circular DNA (cccDNA). Thus, CRISPR/Cas9 is a promising therapeutic strategy for anti-HBV therapy. Double-strand breaks (DSBs) and uncontrolled genomic rearrangements occur inevitably. In this study, we aimed to use base editors to control HBV infection.

METHODS: Base editors precisely instal targeted point mutations without requiring DSBs or donor DNA templates, and without relying on homology-directed repair (HDR) or nonhomologous end joining (NHEJ). Adenine base editors (ABEs) and cytosine base editors (CBEs) catalyse A• T to G •C and C• G to T •A conversions, respectively. In this study, to control HBV replication by modifying and inactivating key HBV genes, recently developed CRISPR/Cas-mediated SpRY-ABE8e and CBE4-max were utilised to falsify and invalidate the ATG initiation codons of the S, Pre-S1, PreS2, C, Pre-C, X, and P genes.

RESULTS: The ATG initiation codons of HBV genes were edited by ABE/CBE. The expected point mutations were successfully introduced, resulting in the simultaneous suppression of HBV antigen expression and replication to varying degrees.

CONCLUSIONS: Our study focused on clearing HBV using base and provided experimental and theoretical evidence for the treatment of chronic HBV infection. Thus, base editing is a potential strategy for curing CHB by permanently inactivating the integrated DNA and cccDNA without using DSBs.},
}

@article {pmid40378975,
year = {2025},
author = {Kumar, V and Nagano, T and Takasuka, TE},
title = {Genome integration and expression of β-glucosidase in Priestia megaterium enhanced poly(3-hydroxybutyrate) production from cellobiose and cellulose.},
journal = {Bioresource technology},
volume = {432},
number = {},
pages = {132681},
doi = {10.1016/j.biortech.2025.132681},
pmid = {40378975},
issn = {1873-2976},
abstract = {Polyhydroxyalkanoates (PHAs) production using cellulosic biomass is a promising way for sustainable manufacturing of bioplastics. Priestia megaterium is an ideal choice as it can use glucose and xylose for PHA production. To further improve the strain for PHA production from cellobiose, we integrate exogenous β-glucosidase (Bgl) from Bacillus sp. GL1 (Bsbgl) in the PHA depolymerase (phaZ1) deletion background (ΔZ1) using CRISPR-Cas. The deletion of phaZ1 in P. megaterium showed a significant improvement in the PHA accumulation whereas BsBgl expression resulted in robust activity and improved growth using cellobiose as a sole carbon source compared to other Bgl targets. To further improve the strain, four native promoters were examined for intracellular BsBgl expression, and the PHA promoter (PphaR) and citrate synthase promoter (Pcitz) showed 2.0- and 4.5-fold higher activities of BsBgl, compared to the xylose promoter (Pxyl). The rate of cellobiose utilization in engineered strains P2 (PphaRBsbgl_ΔZ1) and P4 (PcitzBsbgl_ΔZ1) was improved to 1.6-fold and 2.6-fold, whereas poly(3-hydroxybutyrate) (P3HB) yield for the respective strains was around 3-fold to the wild-type. The strain P2 turned out to be better for cellobiose to PHA production. Further, the strain P2 in a co-culture with cellulolytic Streptomyces sp. SirexAA-E in a consolidated bioprocessing yielded 76 mg of P3HB/ g of carboxymethylcellulose, which is 4.3-times higher than the co-culture with the wild-type. Thus, the present work improved the cellobiose utilization and P3HB accumulation of P. megaterium. The current study paves the way for designing efficient cell factories for cellulosic biomass into bioplastic in the future.},
}

@article {pmid40377871,
year = {2025},
author = {Rahmati, R and Zarimeidani, F and Ghanbari Boroujeni, MR and Sadighbathi, S and Kashaniasl, Z and Saleh, M and Alipourfard, I},
title = {CRISPR-Assisted Probiotic and In Situ Engineering of Gut Microbiota: A Prospect to Modification of Metabolic Disorders.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {40377871},
issn = {1867-1314},
abstract = {The gut microbiota, a substantial group of microorganisms residing in the human body, profoundly impacts various physiological and pathological mechanisms. Recent studies have elucidated the association between gut dysbiosis and multiple organ diseases. Gut microbiota plays a crucial role in maintaining gastrointestinal stability, regulating the immune system and metabolic processes not only within the gastrointestinal tract but also in other organs such as the brain, lungs, and skin. Dysbiosis of the gut microbiota can disrupt biological functioning and contribute to the development of metabolic disorders. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) modules are adaptive immune systems in numerous archaea and bacteria. CRISPR/Cas is a versatile gene-editing tool that enables modification of the genome in live cells, including those within the gut microbiota. This technique has revolutionized gene editing due to its simplicity and effectiveness. It finds extensive applications in diverse scientific arenas, facilitating the functional screening of genomes during various biological processes. Additionally, CRISPR has been instrumental in creating model organisms and cell lines for research purposes and holds great potential for developing personalized medical treatments through precise genetic alterations. This review aims to explore and discuss the possibilities of CRISPR/Cas and the current trends in using this technique for editing gut microbiota genes in various metabolic disorders. By uncovering the valuable potential of CRISPR/Cas in modifying metabolic disorders through the human gut microbiota, we shed light on its promising applications.},
}

@article {pmid40377313,
year = {2025},
author = {Scarrone, M and Turner, D and Dion, M and Tremblay, D and Moineau, S},
title = {In silico and in vitro comparative analysis of 79 Acinetobacter baumannii clinical isolates.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0284924},
doi = {10.1128/spectrum.02849-24},
pmid = {40377313},
issn = {2165-0497},
abstract = {Acinetobacter baumannii is a significant nosocomial bacterial pathogen that poses a substantial infection risk due to its high resistance to antibiotics and ability to survive in hospital environments. In this study, we performed comprehensive in silico and in vitro analyses on 79 A. baumannii clinical isolates from different geographical locations to uncover their genomic and epidemiological characteristics as well as their antibiotic and phage susceptibilities. Our findings revealed considerable genomic diversity among the isolates, as shown by average nucleotide identity (ANI) heat maps, multilocus sequence typing (MLST), and core genome MLST (cgMLST). We identified several international clones known for their high antibiotic resistance and global prevalence. Surprisingly, we also observed that the number of antimicrobial resistance genes (ARGs) was higher in isolates containing CRISPR-Cas systems. Plaque assays with 13 phages indicated that Acinetobacter phages have a narrow host range, with capsule loci (KL) serving as a good indicator of phage-bacteria interactions. The presence of CRISPR-Cas systems and other antiviral defense mechanisms in A. baumannii genomes also appears to play a key role in providing phage resistance, regardless of the phage receptors. We also found that spacers associated with subtypes I-F1 and I-F2 CRISPR-Cas systems predominantly target prophages, suggesting a role in maintaining genomic stability and contributing to phage-bacteria co-evolution. Overall, this study provides a set of highly characterized A. baumannii clinical isolates for future studies on antibiotic-phage-bacteria interactions.IMPORTANCEAcinetobacter baumannii poses a significant challenge to the healthcare system due to its antibiotic resistance and strong survival mechanisms. This study examines a diverse collection of 79 clinical isolates to deepen our understanding of A. baumannii's genetic characteristics and its defense mechanisms against both antibiotics and phages. Genomic analysis revealed globally prevalent, highly resistant clones and uncovered a complex role for CRISPR-Cas systems. Although CRISPR-Cas systems were not widespread among these isolates, they primarily targeted prophages. Additionally, the study emphasizes the importance of capsule types as indicators of phage susceptibility. Together, these findings provide insights into the pathogen's resilience and evolutionary adaptations, potentially guiding future research on infection control strategies and new therapeutic approaches to combat A. baumannii infections.},
}

@article {pmid40377112,
year = {2025},
author = {Kellari, LM and Dalakouras, A and Tsiouri, O and Vletsos, P and Katsaouni, A and Uslu, VV and Papadopoulou, KK},
title = {Cross-kingdom RNAi induced by a beneficial endophytic fungus to its host requires transitivity and amplification of silencing signals.},
journal = {Plant biology (Stuttgart, Germany)},
volume = {27},
number = {4},
pages = {504-514},
doi = {10.1111/plb.70026},
pmid = {40377112},
issn = {1438-8677},
support = {6236//the Hellenic Foundation for Research and Innovation (HFRI)/ ; 7322//Research Committee of UTH/ ; //PRIMA Programme/ ; },
mesh = {*Nicotiana/microbiology/genetics/metabolism ; *Endophytes/physiology ; *Fusarium/physiology/genetics ; *RNA Interference ; Green Fluorescent Proteins/metabolism/genetics ; },
abstract = {Cross-kingdom transfer of small RNA (sRNA) molecules has been identified as a means of communication between plants and interacting microorganisms, but the mechanistic details of this sRNA-based interaction remain elusive. We have previously shown that the beneficial root-colonizing fungus Fusarium solani strain K (FsK) translocates sRNAs to its host, Nicotiana benthamiana (Nb), leading to systemic silencing of a reporter gene. Here, we investigated the mechanistic details of the endophyte-induced systemic silencing using an RNAi sensor system. We inoculated three Nb GFP expressing lines with conidia of an FsK transformant containing a transgene that targets host GFP (FsK-hpGF). The efficiency of silencing mediated by FsK-hpGF was monitored both phenotypically under ultraviolet light as well as quantitatively by RT-qPCR. sRNA sequencing was performed to evaluate the production of sRNAs targeting host GFP. Finally, bisulfite sequencing was used to assess plant GFP methylation levels. We show that the translocated fungal sRNAs induced production of secondary sRNAs, mainly of 22-24-nt in size, with the conspicuous absence of 21-nt sRNAs. Importantly, systemic silencing could not be induced in an RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) CRISPR/Cas knockout background, nor in an intron-containing target gene. Overall, our data show that endophyte-induced silencing in the host requires RDR6-mediated transitivity and amplification of silencing signals. Despite being based on an artificial RNAi sensor system, our observations may reflect a more generalized and so far unexplored facet of cross-kingdom RNAi, with RDR6-based transitivity influencing the way symbionts and pathogens elicit systemic phenotypes in their host plants.},
}

*Nicotiana/microbiology/genetics/metabolism
*Endophytes/physiology
*Fusarium/physiology/genetics
*RNA Interference
Green Fluorescent Proteins/metabolism/genetics

@article {pmid40376763,
year = {2025},
author = {Wang, K and Liu, Z},
title = {Plant synthetic biology-based biofortification, strategies and recent progresses.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.13934},
pmid = {40376763},
issn = {1744-7909},
support = {startup fund//Shenzhen University of Advanced Technology/ ; },
abstract = {Hidden hunger, caused by chronic micronutrient deficiencies, affects billions of people worldwide and remains a critical public health issue despite progress in food production. Biofortification offers a promising solution by enhancing nutrient levels within plant tissues through traditional breeding or advanced biotechnologies. Recent advancements in plant synthetic biology have significantly improved biofortification strategies, enabling precise and targeted nutrient enrichment. This mini-review outlines five core strategies in synthetic biology-based biofortification: overexpression of endogenous biosynthetic genes, introduction of heterologous biosynthetic pathways, expression of nutrient-specific transporters, optimization of transcriptional regulation, and protein (directed) evolution. Vitamin B1 biofortification serves as a primary illustrative example due to its historical importance and ongoing relevance. Recent breakthroughs, particularly from Chinese research teams, are also highlighted. Together, these strategies offer transformative potential for addressing global nutritional challenges through precise, sustainable and innovative plant-based approaches.},
}

@article {pmid40376717,
year = {2025},
author = {Marzluf, JP and Daniela, K and Klein, J and Zehe, C and Leroux, AC},
title = {Utilizing Stable Gene-Edited Knockout Pools for Genetic Screening and Engineering in Chinese Hamster Ovary Cells.},
journal = {Biotechnology journal},
volume = {20},
number = {5},
pages = {e70033},
pmid = {40376717},
issn = {1860-7314},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Cricetinae ; *Genetic Testing/methods ; Fibronectins/genetics ; Genetic Engineering/methods ; },
abstract = {Chinese hamster ovary (CHO) cells are the primary host for biopharmaceutical production. To meet increasing demands for productivity, quality, and complex molecule expression, genetic engineering, particularly clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene knockout (KO), is widely used to optimize host cell performance. However, systematic screening of KO targets remains challenging due to the labor-intensive process of generating and evaluating individual clones. In this study, we present a robust, high-throughput CRISPR workflow using stable KO pools in CHO cells. These pools maintain genetic stability for over 6 weeks, including in multiplexed configurations targeting up to seven genes simultaneously. Compared to clonal approaches, KO pools reduce variability caused by clonal heterogeneity and better reflect the host cell population phenotype. We demonstrate the utility of this approach by reproducing the beneficial phenotypic effects of fibronectin 1 (FN1) KO, specifically prolonged culture duration and improved late-stage viability in fed-batch processes. This workflow enables efficient identification and evaluation of promising KO targets without the need to generate and test large numbers of clones. Overall, screening throughput is increased 2.5-fold and timelines are compressed from 9 to 5 weeks. This provides a scalable, efficient alternative to traditional clonal screening, accelerating discovery for CHO cell line engineering for biopharmaceutical development.},
}

CHO Cells
Animals
Cricetulus
*Gene Knockout Techniques/methods
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Cricetinae
*Genetic Testing/methods
Fibronectins/genetics
Genetic Engineering/methods

@article {pmid40376300,
year = {2025},
author = {Feussner, M and Migur, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Beisel, CL and Weinberg, Z},
title = {Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf007},
pmid = {40376300},
issn = {2633-6693},
abstract = {CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the 'extraneous' CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.},
}

@article {pmid40376084,
year = {2024},
author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA},
title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.10.01.616117},
pmid = {40376084},
issn = {2692-8205},
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a 20-base-pair DNA sequence adjacent to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical and cell-based assays on S. pyogenes Cas9 (Spy Cas9) variants with wide-ranging genome editing efficiencies that differ in PAM binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.},
}

@article {pmid40375877,
year = {2025},
author = {Khamwut, A and Nimnual, J and Chomta, N and Nimsamer, P and Mayuramart, O and Kaewsapsak, P and Pasittungkul, S and Poovorawan, Y and Payungporn, S},
title = {Detection of respiratory syncytial virus based on RT-RPA and CRISPR-Cas12a.},
journal = {Experimental biology and medicine (Maywood, N.J.)},
volume = {250},
number = {},
pages = {10387},
pmid = {40375877},
issn = {1535-3699},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Respiratory Syncytial Virus, Human/isolation & purification/genetics ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human respiratory syncytial virus (hRSV) is one of the most prevalent viruses infecting children globally. In this study, we employed the RT-RPA with CRISPR/Cas12a detection methodology to detect and differentiate RSV-A and RSV-B, particularly in resource-limited settings. The detection limit for RSV-A and RSV-B was approximately 10[2] and 10[3] copies/reaction, respectively. The assay revealed 100% specificity in detecting both RSV-A and RSV-B. Diagnostic accuracy was 90.32 and 93.55% for RSV-A and RSV-B, respectively, compared to RT-qPCR. These data indicate a proficient strategy for RSV screening, demonstrating promise for prospective applications in detecting diverse viral infections.},
}

Humans
*CRISPR-Cas Systems/genetics
*Respiratory Syncytial Virus, Human/isolation & purification/genetics
*Respiratory Syncytial Virus Infections/diagnosis/virology
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40374732,
year = {2025},
author = {Hagman, A and Stenström, O and Carlström, G and Akke, M and Grey, C and Carlquist, M},
title = {Biocatalytic reductive amination with CRISPR-Cas9 engineered yeast.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {16972},
pmid = {40374732},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Amination ; *Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Biocatalysis ; Transaminases/genetics/metabolism ; Chromobacterium/enzymology/genetics ; Alanine/metabolism ; Alanine Transaminase/genetics/metabolism ; Bioreactors ; },
abstract = {Metabolically engineered baker's yeast can be used to produce chiral amines through whole-cell bioconversion of prochiral ketones. This study investigates the modulation of the alanine-pyruvate metabolic node to enhance reductive amination, using the stereoselective conversion of benzylacetone to (S)-1-methyl-3-phenylpropylamine (MPPA) as a model reaction. Chromosomal integration of multiple copies of the promiscuous omega transaminase from Chromobacterium violaceum (cv-ATA) resulted in an active yeast catalyst. Physiological characterization in bioreactors under aerobic batch cultivation revealed that amine production occurred only under post-diauxic growth on ethanol. To reduce native alanine utilization, the endogenous alanine aminotransferase (ALT1) was knocked out and replaced with cv-ATA. To rapidly employ this strategy in other strains, a simple CRISPR/cas9 method for universal gene replacement was developed. The replacement of ALT1 with cv-ATA improved the reaction by 2.6-fold compared to the control strain with intact ALT1. NMR measurements of metabolites originating from [15]N L-alanine and [13]C glucose indicated that pyruvate formation during growth on glucose inhibited amine production. Under optimal conditions, the biocatalytic bioconversion of benzylacetone to MPPA reached a yield of 58%.},
}

*CRISPR-Cas Systems
Amination
*Saccharomyces cerevisiae/genetics/metabolism
*Metabolic Engineering/methods
Biocatalysis
Transaminases/genetics/metabolism
Chromobacterium/enzymology/genetics
Alanine/metabolism
Alanine Transaminase/genetics/metabolism
Bioreactors

@article {pmid40373829,
year = {2025},
author = {Trujillo, E and Angulo, C},
title = {Perspectives on the use of the CRISPR system in plants to improve recombinant therapeutic protein production.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {111-123},
doi = {10.1016/j.jbiotec.2025.05.010},
pmid = {40373829},
issn = {1873-4863},
abstract = {The plant-based system is a promising platform for producing biotherapeutics due to its scalability, cost-effectiveness, and lower risk of contamination by human pathogens. However, several challenges remain, including optimizing yield, stability, functionality, and the immunogenic properties of recombinant proteins. In this context, this review explores the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology to improve the production of recombinant therapeutic proteins in plants. Traditional tools and strategies for plant-based recombinant protein production are discussed, highlighting their limitations and the potential of CRISPR to overcome these boundaries. It delves into the components of the CRISPR-Cas system and its application in optimizing therapeutic protein function and yield. Major strategies include modifying glycosylation patterns to humanize plant-produced proteins, metabolic pathway engineering to increase protein accumulation, and the precise integration of transgenes into specific genomic loci to enhance expression stability and productivity. These advancements demonstrate how CRISPR system can overcome bottlenecks in plant molecular farming and enable the production of high-quality therapeutic proteins. Lastly, future trends and perspectives are examined, emphasizing ongoing innovations and challenges in the field. The review underscores the potential of CRISPR to reshape plant biotechnology and support the growing demand for recombinant therapeutics, offering new avenues for sustainable and efficient protein production systems. KEY MESSAGE: CRISPR technology has the potential to improve plant-based therapeutic protein production by optimizing yield, stability, and humanization, overcoming bottlenecks, and enabling sustainable, efficient systems for recombinant biotherapeutics.},
}

@article {pmid40373688,
year = {2025},
author = {Thakur, RK and Aggarwal, K and Sood, N and Kumar, A and Joshi, S and Jindal, P and Maurya, R and Patel, P and Kurmi, BD},
title = {Harnessing advances in mechanisms, detection, and strategies to combat antimicrobial resistance.},
journal = {The Science of the total environment},
volume = {982},
number = {},
pages = {179641},
doi = {10.1016/j.scitotenv.2025.179641},
pmid = {40373688},
issn = {1879-1026},
abstract = {Antimicrobial resistance (AMR) is a growing global health crisis, threatening the effectiveness of antibiotics and other antimicrobial agents, leading to increased morbidity, mortality, and economic burdens. This review article provides a comprehensive analysis of AMR, beginning with a timeline of antibiotics discovery and the year of first observed resistance. Main mechanisms of AMR in bacteria, fungi, viruses, and parasites are summarized, and the main mechanisms of bacteria are given in detail. Additionally, we discussed in detail methods for detecting AMR, including phenotypic, genotypic, and advanced methods, which are crucial for identifying and monitoring AMR. In addressing AMR mitigation, we explore innovative interventions such as CRISPR-Cas systems, nanotechnology, antibody therapy, artificial intelligence (AI), and the One Health approach. Moreover, we discussed both finished and ongoing clinical trials for AMR. This review emphasizes the urgent need for global action and highlights promising technologies that could shape the future of AMR surveillance and treatment. By integrating interdisciplinary research and emerging clinical insights, this study aims to guide individuals toward impactful solutions in the battle against AMR.},
}

@article {pmid40373119,
year = {2025},
author = {Witte, IP and Lampe, GD and Eitzinger, S and Miller, SM and Berríos, KN and McElroy, AN and King, RT and Stringham, OG and Gelsinger, DR and Vo, PLH and Chen, AT and Tolar, J and Osborn, MJ and Sternberg, SH and Liu, DR},
title = {Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6748},
pages = {eadt5199},
doi = {10.1126/science.adt5199},
pmid = {40373119},
issn = {1095-9203},
mesh = {Humans ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems ; *Mutagenesis, Insertional ; },
abstract = {Programmable gene integration in human cells has the potential to enable mutation-agnostic treatments for loss-of-function genetic diseases and facilitate many applications in the life sciences. CRISPR-associated transposases (CASTs) catalyze RNA-guided DNA integration but thus far demonstrate minimal activity in human cells. Using phage-assisted continuous evolution (PACE), we generated CAST variants with >200-fold average improved integration activity. The evolved CAST system (evoCAST) achieves ~10 to 30% integration efficiencies of kilobase-size DNA cargoes in human cells across 14 tested genomic target sites, including safe harbor loci, sites used for immunotherapy, and genes implicated in loss-of-function diseases, with undetected indels and low levels of off-target integration. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile system for programmable gene integration in living systems.},
}

Humans
*Transposases/genetics/metabolism
*CRISPR-Cas Systems
*Directed Molecular Evolution/methods
*Gene Editing/methods
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems
*Mutagenesis, Insertional