@article {pmid39908088, year = {2025}, author = {Din, NS and Mohd Rani, F and Alattraqchi, AG and Ismail, S and A Rahman, NI and Cleary, DW and Clarke, SC and Yeo, CC}, title = {Whole-genome sequencing of Acinetobacter baumannii clinical isolates from a tertiary hospital in Terengganu, Malaysia (2011-2020), revealed the predominance of the Global Clone 2 lineage.}, journal = {Microbial genomics}, volume = {11}, number = {2}, pages = {}, doi = {10.1099/mgen.0.001345}, pmid = {39908088}, issn = {2057-5858}, mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation & purification/classification ; Malaysia/epidemiology ; *Tertiary Care Centers ; Humans ; *Whole Genome Sequencing ; *Drug Resistance, Multiple, Bacterial/genetics ; *Acinetobacter Infections/microbiology/epidemiology ; *Multilocus Sequence Typing ; *Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Genome, Bacterial ; Male ; beta-Lactamases/genetics ; Adult ; Female ; Middle Aged ; Aged ; Bacterial Proteins/genetics ; Young Adult ; Phylogeny ; Adolescent ; Aged, 80 and over ; Child ; }, abstract = {Carbapenem-resistant Acinetobacter baumannii is recognized by the World Health Organization (WHO) as one of the top priority pathogens. Despite its public health importance, genomic data of clinical isolates from Malaysia remain scarce. In this study, whole-genome sequencing was performed on 126 A. baumannii isolates collected from the main tertiary hospital in the state of Terengganu, Malaysia, over a 10-year period (2011-2020). Antimicrobial susceptibilities determined for 20 antibiotics belonging to 8 classes showed that 77.0% (n=97/126) of the isolates were categorized as multidrug resistant (MDR), with all MDR isolates being carbapenem resistant. Multilocus sequence typing analysis categorized the Terengganu A. baumannii clinical isolates into 34 Pasteur and 44 Oxford sequence types (STs), with ST2Pasteur of the Global Clone 2 lineage identified as the dominant ST (n=76/126; 60.3%). The ST2Pasteur isolates could be subdivided into six Oxford STs with the majority being ST195Oxford (n=35) and ST208Oxford (n=17). Various antimicrobial resistance genes were identified with the bla OXA-23-encoded carbapenemase being the predominant acquired carbapenemase gene (n=90/126; 71.4%). Plasmid-encoded rep genes were identified in nearly all (n=122/126; 96.8%) of the isolates with the majority being Rep_3 family (n=121). Various virulence factors were identified, highlighting the pathogenic nature of this bacterium. Only 14/126 (11.1%) of the isolates were positive for the carriage of CRISPR-Cas arrays with none of the prevalent ST2Pasteur isolates harbouring them. This study provided a genomic snapshot of the A. baumannii isolates obtained from a single tertiary healthcare centre in Malaysia over a 10-year period and showed the predominance of a single closely related ST2Pasteur lineage, indicating the entrenchment of this clone in the hospital.}, }
@article {pmid39907946, year = {2025}, author = {Rachappanavar, V}, title = {Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {204}, pmid = {39907946}, issn = {1573-4978}, mesh = {*Plant Dormancy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Seeds/genetics ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant/genetics ; Germination/genetics ; }, abstract = {Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.}, }
@article {pmid39907302, year = {2025}, author = {Talat, A and Khan, AU}, title = {From supplements to superbugs: how probiotic patent gaps drive antimicrobial resistance and the CRISPR-Cas solutions.}, journal = {Pharmaceutical patent analyst}, volume = {}, number = {}, pages = {1-3}, doi = {10.1080/20468954.2025.2459592}, pmid = {39907302}, issn = {2046-8962}, }
@article {pmid39906792, year = {2025}, author = {Ali Agha, ASA and Al-Samydai, A and Aburjai, T}, title = {New frontiers in CRISPR: Addressing antimicrobial resistance with Cas9, Cas12, Cas13, and Cas14.}, journal = {Heliyon}, volume = {11}, number = {2}, pages = {e42013}, pmid = {39906792}, issn = {2405-8440}, abstract = {BACKGROUND: The issue of antimicrobial resistance (AMR) poses a major challenge to global health, evidenced by alarming mortality predictions and the diminishing efficiency of conventional antimicrobial drugs. The CRISPR-Cas system has proven to be a powerful tool in addressing this challenge. It originated from bacterial adaptive immune mechanisms and has gained significant recognition in the scientific community.
OBJECTIVES: This review aims to explore the applications of CRISPR-Cas technologies in combating AMR, evaluating their effectiveness, challenges, and potential for integration into current antimicrobial strategies.
METHODS: We conducted a comprehensive review of recent literature from databases such as PubMed and Web of Science, focusing on studies that employ CRISPR-Cas technologies against AMR.
CONCLUSIONS: CRISPR-Cas technologies offer a transformative approach to combat AMR, with potential applications that extend beyond traditional antimicrobial strategies. Integrating these technologies with existing methods could significantly enhance our ability to manage and potentially reverse the growing problem of antimicrobial resistance. Future research should address technical and ethical barriers to facilitate safe and effective clinical and environmental applications. This review underscores the necessity for interdisciplinary collaboration and international cooperation to harness the full potential of CRISPR-Cas technologies in the fight against superbugs.}, }
@article {pmid39906170, year = {2025}, author = {Vu, TV and Nguyen, NT and Kim, J and Vu, MH and Song, YJ and Tran, MT and Sung, YW and Kim, JY}, title = {Enhancing CRISPR-Cas-based gene targeting in tomato using a dominant-negative ku80.}, journal = {Horticulture research}, volume = {12}, number = {2}, pages = {uhae294}, pmid = {39906170}, issn = {2662-6810}, abstract = {The CRISPR-Cas-based gene targeting (GT) method has enabled precise modifications of genomic DNA ranging from single base to several kilobase scales through homologous recombination (HR). In plant somatic cells, canonical non-homologous end-joining (cNHEJ) is the predominant mechanism for repairing double-stranded breaks (DSBs), thus limiting the HR-mediated GT. In this study, we implemented an approach to shift the repair pathway preference toward HR by using a dominant-negative ku80 mutant protein (KUDN) to disrupt the initiation of cNHEJ. The employment of KUDN conferred a 1.71- to 3.55-fold improvement in GT efficiency at the callus stage. When we screened transformants, there was a more remarkable increase in GT efficiency, ranging from 1.62- to 9.84-fold, at two specific tomato loci, SlHKT1;2 and SlEPSPS1. With practical levels of efficiency, this enhanced KUDN-based GT tool successfully facilitated a 9-bp addition at an additional locus, SlCAB13. These findings provide another promising method for more efficient and precise plant breeding.}, }
@article {pmid39906167, year = {2025}, author = {Bilal, M and Geng, J and Chen, L and García-Caparros, P and Hu, T}, title = {Genome editing for grass improvement and future agriculture.}, journal = {Horticulture research}, volume = {12}, number = {2}, pages = {uhae293}, pmid = {39906167}, issn = {2662-6810}, abstract = {Grasses, including turf and forage, cover most of the earth's surface; predominantly important for land, water, livestock feed, soil, and water conservation, as well as carbon sequestration. Improved production and quality of grasses by modern molecular breeding is gaining more research attention. Recent advances in genome-editing technologies are helping to revolutionize plant breeding and also offering smart and efficient acceleration on grass improvement. Here, we reviewed all recent researches using (CRISPR)/CRISPR-associated protein (Cas)-mediated genome editing tools to enhance the growth and quality of forage and turf grasses. Furthermore, we highlighted emerging approaches aimed at advancing grass breeding program. We assessed the CRISPR-Cas effectiveness, discussed the challenges associated with its application, and explored future perspectives primarily focusing on turf and forage grasses. Despite the promising potential of genome editing in grasses, its current efficiency remains limited due to several bottlenecks, such as the absence of comprehensive reference genomes, the lack of efficient gene delivery tools, unavailability of suitable vector and delivery for grass species, high polyploidization, and multiple homoeoalleles, etc. Despite these challenges, the CRISPR-Cas system holds great potential to fully harness its benefits in grass breeding and genetics, aiming to improve and sustain the quantity and quality of turf and forage grasses.}, }
@article {pmid39905471, year = {2025}, author = {Ye, T and Ding, W and An, Z and Zhang, H and Wei, X and Xu, J and Liu, H and Fang, H}, title = {Increased distribution of carbon metabolic flux during de novo cytidine biosynthesis via attenuation of the acetic acid metabolism pathway in Escherichia coli.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {36}, pmid = {39905471}, issn = {1475-2859}, support = {31860020//National Natural Science Foundation of China/ ; 022004000010//Ningxia Hui Autonomous Region Youth Top Talent Training Project/ ; No. 2020//The Helanshan Scholars Program of Ningxia University/ ; 2023AAC02030//Natural Science Foundation of Ningxia, China/ ; }, mesh = {*Acetic Acid/metabolism ; *Escherichia coli/metabolism/genetics ; *Carbon/metabolism ; *Cytidine/metabolism ; Metabolic Engineering/methods ; Fermentation ; Escherichia coli Proteins/metabolism/genetics ; Metabolic Networks and Pathways ; Glucose/metabolism ; CRISPR-Cas Systems ; Citric Acid Cycle ; }, abstract = {Acetic acid, a by-product of cytidine synthesis, competes for carbon flux from central metabolism, which may be directed either to the tricarboxylic acid (TCA) cycle for cytidine synthesis or to overflow metabolites, such as acetic acid. In Escherichia coli, the acetic acid synthesis pathway, regulated by the poxB and pta genes, facilitates carbon consumption during cytidine production. To mitigate carbon source loss, the CRISPR-Cas9 gene-editing technique was employed to knock out the poxB and pta genes in E. coli, generating the engineered strains K12ΔpoxB and K12ΔpoxBΔpta. After 39 h of fermentation in 500 mL shake flasks, the cytidine yields of strains K12ΔpoxB and K12ΔpoxBΔpta were 1.91 ± 0.04 g/L and 18.28 ± 0.22 g/L, respectively. Disruption of the poxB and pta genes resulted in reduced acetic acid production and glucose consumption. Transcriptomic and metabolomic analyses revealed that impairing the acetic acid metabolic pathway in E. coli effectively redirected carbon flux toward cytidine biosynthesis, yielding a 5.26-fold reduction in acetate metabolism and an 11.56-fold increase in cytidine production. These findings provide novel insights into the influence of the acetate metabolic pathway on cytidine biosynthesis in E. coli.}, }
@article {pmid39905452, year = {2025}, author = {Abbasi, AF and Asim, MN and Dengel, A}, title = {Transitioning from wet lab to artificial intelligence: a systematic review of AI predictors in CRISPR.}, journal = {Journal of translational medicine}, volume = {23}, number = {1}, pages = {153}, pmid = {39905452}, issn = {1479-5876}, mesh = {*Artificial Intelligence ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {The revolutionary CRISPR-Cas9 system leverages a programmable guide RNA (gRNA) and Cas9 proteins to precisely cleave problematic regions within DNA sequences. This groundbreaking technology holds immense potential for the development of targeted therapies for a wide range of diseases, including cancers, genetic disorders, and hereditary diseases. CRISPR-Cas9 based genome editing is a multi-step process such as designing a precise gRNA, selecting the appropriate Cas protein, and thoroughly evaluating both on-target and off-target activity of the Cas9-gRNA complex. To ensure the accuracy and effectiveness of CRISPR-Cas9 system, after the targeted DNA cleavage, the process requires careful analysis of the resultant outcomes such as indels and deletions. Following the success of artificial intelligence (AI) in various fields, researchers are now leveraging AI algorithms to catalyze and optimize the multi-step process of CRISPR-Cas9 system. To achieve this goal AI-driven applications are being integrated into each step, but existing AI predictors have limited performance and many steps still rely on expensive and time-consuming wet-lab experiments. The primary reason behind low performance of AI predictors is the gap between CRISPR and AI fields. Effective integration of AI into multi-step CRISPR-Cas9 system demands comprehensive knowledge of both domains. This paper bridges the knowledge gap between AI and CRISPR-Cas9 research. It offers a unique platform for AI researchers to grasp deep understanding of the biological foundations behind each step in the CRISPR-Cas9 multi-step process. Furthermore, it provides details of 80 available CRISPR-Cas9 system-related datasets that can be utilized to develop AI-driven applications. Within the landscape of AI predictors in CRISPR-Cas9 multi-step process, it provides insights of representation learning methods, machine and deep learning methods trends, and performance values of existing 50 predictive pipelines. In the context of representation learning methods and classifiers/regressors, a thorough analysis of existing predictive pipelines is utilized for recommendations to develop more robust and precise predictive pipelines.}, }
@article {pmid39905300, year = {2025}, author = {Sun, W and Zhang, J and Li, S and Fu, W and Liu, Y and Liu, M and Dong, J and Zhao, X and Li, X}, title = {TAB2 deficiency induces dilated cardiomyopathy by promoting mitochondrial calcium overload in human iPSC-derived cardiomyocytes.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {31}, number = {1}, pages = {42}, pmid = {39905300}, issn = {1528-3658}, support = {82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 82000352//National Natural Science Foundation of China/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; 2020M672295//China Postdoctoral Science Foundation/ ; }, mesh = {Humans ; *Myocytes, Cardiac/metabolism ; *Cardiomyopathy, Dilated/metabolism/genetics/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; *Calcium/metabolism ; Cell Differentiation ; Mitochondria/metabolism ; Gene Knockout Techniques ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediary protein that links Tumor necrosis factor receptor 1 (TNFR1) and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 frameshift mutations have been linked to dilated cardiomyopathy (DCM), while the exact mechanism needs further investigation.
METHODS: In this study, we generated a TAB2 compound heterozygous knockout cell line in induced pluripotent stem cells (iPSCs) derived from a healthy individual using CRISPR/Cas9 technology. IPSCs are not species-dependent, are readily accessible, and raise fewer ethical concerns.
RESULTS: TAB2 disruption had no impact on the cardiac differentiation of iPSCs and led to confirmed TAB2 deficiency in human iPSC-derived cardiomyocytes (hiPSC-CMs). TAB2-deficient hiPSC-CMs were found to develop phenotypic features of DCM, such as distorted sarcomeric ultrastructure, decreased contractility and energy production, and mitochondrial damage at day 30 post differentiation. Paradoxically, TAB2 knockout cell lines showed abnormal calcium handling after 40 days, later than reduced contractility, suggesting that the main cause of impaired contractility was abnormal energy production due to mitochondrial damage. As early as day 25, TAB2 knockout cardiomyocytes showed significant mitochondrial calcium overload, which can lead to mitochondrial damage. Furthermore, TAB2 knockout activated receptor-interacting protein kinase 1 (RIPK1), leading to an increase in mitochondrial calcium uniporter (MCU) expression, thereby augmenting the uptake of mitochondrial calcium ions. Finally, the application of the RIPK1 inhibitor Nec-1s prevents the progression of these phenotypes.
CONCLUSIONS: In summary, TAB2 abatement cardiomyocytes mimic dilated cardiomyopathy in vitro. This finding emphasizes the importance of using a human model to study the underlying mechanisms of this specific disease. More importantly, the discovery of a unique pathogenic pathway introduces a new notion for the future management of dilated cardiomyopathy.}, }
@article {pmid39905017, year = {2025}, author = {Moore, MM and Wekhande, S and Issner, R and Collins, A and Cruz, AJ and Liu, YV and Javed, N and Casaní-Galdón, S and Buenrostro, JD and Epstein, CB and Mattei, E and Doench, JG and Bernstein, BE and Shoresh, N and Najm, FJ}, title = {Multi-locus CRISPRi targeting with a single truncated guide RNA.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1357}, pmid = {39905017}, issn = {2041-1723}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Enhancer Elements, Genetic/genetics ; Genetic Loci ; Histones/metabolism/genetics ; }, abstract = {A critical goal in functional genomics is evaluating which non-coding elements contribute to gene expression, cellular function, and disease. Functional characterization remains a challenge due to the abundance and complexity of candidate elements. Here, we develop a CRISPRi-based approach for multi-locus screening of putative transcription factor binding sites with a single truncated guide. A truncated guide with hundreds of sequence match sites can reliably disrupt enhancer activity, which expands the targeting scope of CRISPRi while maintaining repressive efficacy. We screen over 13,000 possible CTCF binding sites with 24 guides at 10 nucleotides in spacer length. These truncated guides direct CRISPRi-mediated deposition of repressive H3K9me3 marks and disrupt transcription factor binding at most sequence match target sites. This approach can be a valuable screening step for testing transcription factor binding motifs or other repeated genomic sequences and is easily implemented with existing tools.}, }
@article {pmid39904990, year = {2025}, author = {Liu, Y and Wang, L and Zhang, Q and Fu, P and Zhang, L and Yu, Y and Zhang, H and Zhu, H}, title = {Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1335}, pmid = {39904990}, issn = {2041-1723}, mesh = {*Cryoelectron Microscopy ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *DNA/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Bacterial Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Mutation ; Protein Binding ; DNA Cleavage ; Protein Domains ; }, abstract = {Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) system is one of the most extensively studied RNA-guided adaptive immune systems in prokaryotes, providing defense against foreign genetic elements. Unlike the previously characterized Cas3 nuclease, which exhibits progressive DNA cleavage in the typical type I-E system, a recently identified HNH-comprising Cascade system enables precise DNA cleavage. Here, we present several near-atomic cryo-electron microscopy (cryo-EM) structures of the Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex, both in its DNA-bound and unbound states. Our analysis reveals extensive interactions between the HNH domain and adjacent subunits, including Cas6 and Cas11, with mutations in these key interactions significantly impairing enzymatic activity. Upon DNA binding, the Cas5-HNH/Cascade complex adopts a more compact conformation, with subunits converging toward the center of nuclease, leading to its activation. Notably, we also find that divalent ions such as zinc, cobalt, and nickel down-regulate enzyme activity by destabilizing the Cascade complex. Together, these findings offer structural insights into the assembly and activation of the Cas5-HNH/Cascade complex.}, }
@article {pmid39904298, year = {2025}, author = {Zhong, WJ and Yang, WG and Zhang, Y and Li, T and Su, ML and Yuan, R and Xu, S and Liang, WB}, title = {An electrochemiluminescence strategy with proximity ligation triggered multiple catalytic hairpin assembly induced CRISPR/Cas 12a system for analysis of paraquat.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {164}, number = {}, pages = {108915}, doi = {10.1016/j.bioelechem.2025.108915}, pmid = {39904298}, issn = {1878-562X}, abstract = {Paraquat (PQ) as a widely used non-selective herbicides has gained attention in agricultural residue detection and food safety. Herein, a novel quantitative analysis approach for PQ was proposed based on a novel kind of aggregation-induced emission electrochemiluminescence (AIECL) emitters, tetraphenylethylene-luminol (TPE-L) with a small molecule-induced multiple catalytic hairpin assembly (CHA) amplification strategy, the competitive immune reaction and CRISPR/Cas12a system. The target molecule PQ is introduced into a signal cycle, and auxiliary sensitization cycles are constructed by virtue of the cleavage characteristics of the CRISPR/Cas12a system, which realized the multiple utilization of the target by using both cis- and trans-cleavage activities. In addition, the new multiple CHA amplification strategy was attributed to cross-catalytic hairpin assembly caused by the products of the CHA cycle as the initiator chain of the next CHA cycle, realizing the efficient utilization of cyclic products and producing high-efficiency signal amplification. Thus, the ECL biosensor for ultrasensitive analysis of PQ was successfully constructed with a limit of detection of 0.7 pg/mL. Importantly, it could be easily-extended to other small molecules simply by replacing paired antibodies, providing prospects in agricultural residue detection, food safety and related medical applications.}, }
@article {pmid39903512, year = {2025}, author = {Han, HJ and Yu, D and Yu, J and Kim, J and Do Heo, W and Tark, D and Kang, SM}, title = {Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication.}, journal = {The Journal of general virology}, volume = {106}, number = {2}, pages = {}, pmid = {39903512}, issn = {1465-2099}, mesh = {*Porcine epidemic diarrhea virus/genetics/physiology ; *Virus Replication/drug effects ; Animals ; Chlorocebus aethiops ; *CRISPR-Cas Systems ; Swine ; Vero Cells ; RNA, Viral/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Swine Diseases/virology ; RNA-Dependent RNA Polymerase/genetics/metabolism ; Antiviral Agents/pharmacology ; }, abstract = {Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.}, }
@article {pmid39863179, year = {2025}, author = {Zheng, Y and Wang, S and Deng, Y and Hu, P and Xue, Q and Li, J and Lei, L and Chan, Z and Yang, J and Peng, W}, title = {Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering.}, journal = {Bioresource technology}, volume = {419}, number = {}, pages = {132116}, doi = {10.1016/j.biortech.2025.132116}, pmid = {39863179}, issn = {1873-2976}, mesh = {*Kluyveromyces/genetics/metabolism ; *Chymosin/metabolism/genetics ; *CRISPR-Cas Systems ; *Recombinant Proteins/genetics/metabolism ; Animals ; Cattle ; Fermentation ; }, abstract = {As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.}, }
@article {pmid39854604, year = {2025}, author = {He, X and Sun, Y and Ma, H}, title = {ParSite is a multicolor DNA labeling system that allows for simultaneous imaging of triple genomic loci in living cells.}, journal = {PLoS biology}, volume = {23}, number = {1}, pages = {e3003009}, doi = {10.1371/journal.pbio.3003009}, pmid = {39854604}, issn = {1545-7885}, mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Human ; DNA/genetics/metabolism ; Genetic Loci ; Cell Line, Tumor ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; DNA Breaks, Double-Stranded ; Staining and Labeling/methods ; }, abstract = {The organization of the human genome in space and time is critical for transcriptional regulation and cell fate determination. However, robust methods for tracking genome organization or genomic interactions over time in living cells are lacking. Here, we developed a multicolor DNA labeling system, ParSite, to simultaneously track triple genomic loci in the U2OS cells. The tricolor ParSite system is derived from the T. thermophilus ParB/ParSc (TtParB/ParSc) system by rational design. We mutated the interface between TtParB and ParSc and generated a new pair of TtParBm and ParSm for genomic DNA labeling. The insertions of 16 base-pair palindromic ParSc and ParSm into genomic loci allow dual-color DNA imaging in living cells. A pair of genomic loci labeled by ParSite could be colocalized with p53-binding protein 1 (53BP1) in response to CRISPR/Cas9-mediated double-strand breaks (DSBs). The ParSite permits tracking promoter and terminator dynamics of the APP gene, which spans 290 kilobases in length. Intriguingly, the hybrid ParS (ParSh) of half-ParSc and half-ParSm enables for the visualization of a third locus independent of ParSc or ParSm. We simultaneously labeled 3 loci with a genomic distance of 36, 89, and 352 kilobases downstream the C3 repeat locus, respectively. In sum, the ParSite is a robust DNA labeling system for tracking multiple genomic loci in space and time in living cells.}, }
@article {pmid39762408, year = {2025}, author = {Kim, DH and Choi, SH and Sung, JJ and Kim, S and Yi, H and Park, S and Park, CW and Oh, YW and Lee, J and Kim, DS and Kim, JH and Park, CY and Kim, DW}, title = {Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs.}, journal = {Experimental & molecular medicine}, volume = {57}, number = {1}, pages = {184-192}, pmid = {39762408}, issn = {2092-6413}, support = {2022R1A2C2091165//National Research Foundation of Korea (NRF)/ ; 2022R1A2C1091800//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Hemophilia A/therapy/genetics ; *Factor VIII/genetics/metabolism ; Humans ; Animals ; Mice ; *Genetic Therapy/methods ; Gene Editing/methods ; Peptide Elongation Factor 1/genetics/metabolism ; Disease Models, Animal ; Endothelial Cells/metabolism ; Genetic Vectors/genetics ; Dependovirus/genetics ; Genetic Loci ; Cell Differentiation ; CRISPR-Cas Systems ; Mutation ; }, abstract = {Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.}, }
@article {pmid39760520, year = {2025}, author = {Zhao, Z and Zhang, H and Li, W and Wang, Y and Wang, Y and Yang, H and Yin, L and Liu, X}, title = {Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing.}, journal = {Journal of materials chemistry. B}, volume = {13}, number = {6}, pages = {1991-2002}, doi = {10.1039/d4tb02009j}, pmid = {39760520}, issn = {2050-7518}, mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Peptides/chemistry/pharmacology ; Guanidine/chemistry ; Cytosol/metabolism ; Protein Conformation, alpha-Helical ; }, abstract = {Intracellular delivery of proteins has attracted significant interest in biological research and cancer treatment, yet it continues to face challenges due to the lack of effective delivery approaches. Herein, we developed an efficient strategy via cationic α-helical polypeptide-mediated anionic proprotein delivery. The protein was reversibly modified with adenosine triphosphate via dynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated α-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid α-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including α-chymotrypsin, bovine serum albumin, ribonuclease A, cytochrome C, saporin, horseradish peroxidase, β-galactosidase, and anti-phospho-Akt, into cancer cells. More importantly, it enabled efficient delivery of CRISPR-Cas9 ribonucleoproteins to elicit robust polo-like kinase 1 genome editing for inhibiting cancer cell growth. This rationally designed protein delivery system may benefit the development of intracellular protein-based cancer therapy.}, }
@article {pmid39727169, year = {2025}, author = {Pujar, A and Pathania, A and Hopper, C and Pandi, A and Calderón, CR and Függer, M and Nowak, T and Kushwaha, M}, title = {Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkae1256}, pmid = {39727169}, issn = {1362-4962}, support = {//igicosme working group HicDiesMeus/ ; COMBACT//Ile-de-France (IdF) region's DIM-RFSI/ ; BACON//NS2I CNRS/ ; DEPEC MODE//Université Paris-Saclay's STIC department/ ; PHEMO//INRAE's MICA department/ ; ANR-21-CE48-0003//Agence Nationale de la Recherche/ ; }, mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; Bacteria/genetics/metabolism ; Microbial Consortia/genetics ; Synthetic Biology/methods ; Escherichia coli/genetics/metabolism ; }, abstract = {Coordinated actions of cells in microbial communities and multicellular organisms enable them to perform complex tasks otherwise difficult for single cells. This has inspired biological engineers to build cellular consortia for larger circuits with improved functionalities while implementing communication systems for coordination among cells. Here, we investigate the signalling dynamics of a phage-mediated synthetic DNA messaging system and couple it with CRISPR interference to build distributed circuits that perform logic gate operations in multicellular bacterial consortia. We find that growth phases of both sender and receiver cells, as well as resource competition between them, shape communication outcomes. Leveraging the easy programmability of DNA messages, we build eight orthogonal signals and demonstrate that intercellular CRISPRi (i-CRISPRi) regulates gene expression across cells. Finally, we multiplex the i-CRISPRi system to implement several multicellular logic gates that involve up to seven cells and take up to three inputs simultaneously, with single- and dual-rail encoding: NOT, YES, AND and AND-AND-NOT. The communication system developed here lays the groundwork for implementing complex biological circuits in engineered bacterial communities, using phage signals for communication.}, }
@article {pmid39676667, year = {2025}, author = {Muench, P and Fiumara, M and Southern, N and Coda, D and Aschenbrenner, S and Correia, B and Gräff, J and Niopek, D and Mathony, J}, title = {A modular toolbox for the optogenetic deactivation of transcription.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkae1237}, pmid = {39676667}, issn = {1362-4962}, support = {101041570//European Union/ ; 453202693//German Research Foundation/ ; //Executive Agency/ ; //Aventis Foundation/ ; CoG 101043457//ERC/SERI/ ; //Vallee Foundation/ ; //HFSP/ ; 310 030_197752/SNSF_/Swiss National Science Foundation/Switzerland ; CoG 101043457//SERI/ ; }, mesh = {*Optogenetics/methods ; Humans ; *Transcriptional Activation ; HEK293 Cells ; *Light ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Herpes Simplex Virus Protein Vmw65/genetics/metabolism ; Transcription, Genetic ; Gene Expression Regulation ; Animals ; }, abstract = {Light-controlled transcriptional activation is a commonly used optogenetic strategy that allows researchers to regulate gene expression with high spatiotemporal precision. The vast majority of existing tools are, however, limited to light-triggered induction of gene expression. Here, we inverted this mode of action and created optogenetic systems capable of efficiently terminating transcriptional activation in response to blue light. First, we designed highly compact regulators by photo-controlling the VP16 (pcVP16) transactivation peptide. Then, applying a two-hybrid strategy, we engineered LOOMINA (light off-operated modular inductor of transcriptional activation), a versatile transcriptional control platform for mammalian cells that is compatible with various effector proteins. Leveraging the flexibility of CRISPR systems, we combined LOOMINA with dCas9 to control transcription with blue light from endogenous promoters with exceptionally high dynamic ranges in multiple cell lines. Functionally and mechanistically, the versatile LOOMINA platform and the exceptionally compact pcVP16 transactivator represent valuable additions to the optogenetic repertoire for transcriptional regulation.}, }
@article {pmid39551873, year = {2025}, author = {Krishnamoorthy, V and Daly, J and Kim, J and Piatnitca, L and Yuen, KA and Kumar, B and Taherzadeh Ghahfarrokhi, M and Bui, TQT and Azadi, P and Vu, LP and Wisnovsky, S}, title = {The glycosyltransferase ST3GAL4 drives immune evasion in acute myeloid leukemia by synthesizing ligands for the glyco-immune checkpoint receptor Siglec-9.}, journal = {Leukemia}, volume = {39}, number = {2}, pages = {346-359}, pmid = {39551873}, issn = {1476-5551}, mesh = {Humans ; *Leukemia, Myeloid, Acute/immunology/metabolism/genetics/pathology ; *Sialyltransferases/metabolism/genetics ; *Antigens, CD/metabolism/genetics ; Ligands ; Immune Evasion ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics ; beta-Galactoside alpha-2,3-Sialyltransferase ; Antigens, Differentiation, B-Lymphocyte/metabolism/genetics ; CRISPR-Cas Systems ; Tumor Escape ; }, abstract = {Immunotherapy has demonstrated promise as a treatment for acute myeloid leukemia (AML). However, there is still an urgent need to identify new molecules that inhibit the immune response to AML. Most prior research in this area has focused on protein-protein interaction interfaces. While carbohydrates also regulate immune recognition, the role of cell-surface glycans in driving AML immune evasion is comparatively understudied. The Siglecs, for example, are an important family of inhibitory, glycan-binding signaling receptors that have emerged as prime targets for cancer immunotherapy in recent years. In this study, we find that AML cells express ligands for the receptor Siglec-9 at high levels. Integrated CRISPR genomic screening and clinical bioinformatic analysis identified ST3GAL4 as a potential driver of Siglec-9 ligand expression in AML. Depletion of ST3GAL4 by CRISPR-Cas9 knockout (KO) dramatically reduced the expression of Siglec-9 ligands in AML cells. Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed that Siglec-9 primarily binds N-linked sialoglycans on these cell types. Finally, we found that ST3GAL4 KO enhanced the sensitivity of AML cells to phagocytosis by Siglec-9-expressing macrophages. This work reveals a novel axis of immune evasion and implicates ST3GAL4 as a possible target for immunotherapy in AML.}, }
@article {pmid39307530, year = {2025}, author = {Wang, L and O'Conner, S and Tanvir, R and Zheng, W and Cothron, S and Towery, K and Bi, H and Ellison, EE and Yang, B and Voytas, DF and Li, L}, title = {CRISPR/Cas9-based editing of NF-YC4 promoters yields high-protein rice and soybean.}, journal = {The New phytologist}, volume = {245}, number = {5}, pages = {2103-2116}, doi = {10.1111/nph.20141}, pmid = {39307530}, issn = {1469-8137}, support = {RCZK202468//Shihezi University/ ; //Iowa Soybean Association/ ; //Amfora, Inc./ ; 0951170//National Science Foundation/ ; 2238942//National Science Foundation/ ; CZ001616//Tianchiyingcai Foundation/ ; }, mesh = {*Oryza/genetics ; *Glycine max/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Promoter Regions, Genetic/genetics ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CCAAT-Binding Factor/genetics/metabolism ; Base Sequence ; Plant Leaves/genetics/metabolism ; Seeds/genetics ; }, abstract = {Genome editing is a revolution in biotechnology for crop improvement with the final product lacking transgenes. However, most derived traits have been generated through edits that create gene knockouts. Our study pioneers a novel approach, utilizing gene editing to enhance gene expression by eliminating transcriptional repressor binding motifs. Building upon our prior research demonstrating the protein-boosting effects of the transcription factor NF-YC4, we identified conserved motifs targeted by RAV and WRKY repressors in the NF-YC4 promoters from rice (Oryza sativa) and soybean (Glycine max). Leveraging CRISPR/Cas9 technology, we deleted these motifs, resulting in reduced repressor binding and increased NF-YC4 expression. This strategy led to increased protein content and reduced carbohydrate levels in the edited rice and soybean plants, with rice exhibiting up to a 68% increase in leaf protein and a 17% increase in seed protein, and soybean showing up to a 25% increase in leaf protein and an 11% increase in seed protein. Our findings provide a blueprint for enhancing gene expression through precise genomic deletions in noncoding sequences, promising improved agricultural productivity and nutritional quality.}, }
@article {pmid39902289, year = {2024}, author = {Peach, LJ and Zhang, H and Weaver, BP and Boedicker, JQ}, title = {Assessing spacer acquisition rates in E. coli type I-E CRISPR arrays.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1498959}, pmid = {39902289}, issn = {1664-302X}, abstract = {CRISPR/Cas is an adaptive defense mechanism protecting prokaryotes from viruses and other potentially harmful genetic elements. Through an adaptation process, short "spacer" sequences, captured from these elements and incorporated into a CRISPR array, provide target specificity for the immune response. CRISPR arrays and array expansion are also central to many emerging biotechnologies. The rates at which spacers integrate into native arrays within bacterial populations have not been quantified. Here, we measure naïve spacer acquisition rates in Escherichia coli Type I-E CRISPR, identify factors that affect these rates, and model this process fundamental to CRISPR/Cas defense. Prolonged Cas1-Cas2 expression produced fewer new spacers per cell on average than predicted by the model. Subsequent experiments revealed that this was due to a mean fitness reduction linked to array-expanded populations. In addition, the expression of heterologous non-homologous end-joining DNA-repair genes was found to augment spacer acquisition rates, translating to enhanced phage infection defense. Together, these results demonstrate the impact of intracellular factors that modulate spacer acquisition and identify an intrinsic fitness effect associated with array-expanded populations.}, }
@article {pmid39902056, year = {2025}, author = {Sumanto Marpaung, DS and Yap Sinaga, AO and Damayanti, D and Taharuddin, T and Gumaran, S}, title = {Current biosensing strategies based on in vitro T7 RNA polymerase reaction.}, journal = {Biotechnology notes (Amsterdam, Netherlands)}, volume = {6}, number = {}, pages = {59-66}, pmid = {39902056}, issn = {2665-9069}, abstract = {Recently, a unique behavior of T7 RNA polymerase has expanded its functionality as a biosensing platform. Various biosensors utilizing T7 RNA polymerase, combined with fluorescent aptamers, electrochemical probes, or CRISPR/Cas systems, have been developed to detect analytes, including nucleic acids and non-nucleic acid target, with high specificity and low detection limits. Each approach demonstrates unique strengths, such as real-time monitoring and minimal interference, but also presents challenges in stability, cost, and reaction optimization. This review provides an overview of T7 RNA polymerase's role in biosensing technology, highlighting its potential to advance diagnostics and molecular detection in diverse fields.}, }
@article {pmid39902050, year = {2024}, author = {Huai, G and Wang, Y and Du, J and Cheng, Z and Xie, Y and Zhou, J and Tang, H and Jiang, Y and Xing, X and Deng, S and Pan, D}, title = {The generation and evaluation of TKO/hCD55/hTM/hEPCR gene-modified pigs for clinical organ xenotransplantation.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1488552}, pmid = {39902050}, issn = {1664-3224}, mesh = {Animals ; *Transplantation, Heterologous ; Humans ; *Animals, Genetically Modified ; Swine ; *Galactosyltransferases/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Membrane Cofactor Protein/genetics/metabolism ; Thrombomodulin/genetics ; Gene Knockout Techniques ; Gene Editing/methods ; Endothelial Cells/metabolism/immunology ; CRISPR-Cas Systems ; Immunoglobulin M/immunology ; CD55 Antigens/genetics ; Mixed Function Oxygenases ; }, abstract = {INTRODUCTION: Genetically edited pigs, modified using CRISPR-Cas9 technology, hold promise as potential sources for xenotransplantation. However, the optimal combination of genetic modifications and their expression levels for initial clinical trials remains unclear. This study investigates the generation of TKO/hCD55/hTM/hEPCR (6GE) pigs and evaluates their compatibility with human immune and coagulation systems.
METHODS: The 6GE pigs were generated through iterative genome editing and F1 generation breeding. Genotyping, flow cytometry, and immunohistochemistry confirmed the knockout of GGTA1, CMAH, and B4GALNT2. Expression levels of human genes (hCD55, hTM, hEPCR) were quantified. In vitro assays using aortic endothelial cells (pAECs) from 6GE pigs assessed human serum IgM and IgG binding, complement cytotoxicity, and thrombin-antithrombin (TAT) complex levels. Blood from gene-edited pigs was used for pathophysiological analysis.
RESULTS: Complete knockout of GGTA1, CMAH, and B4GALNT2 was confirmed in 6GE pigs. The expression of hCD55 and hTM was approximately seven and thirteen times higher than in humans, respectively, while hEPCR levels were comparable to those in humans. In vitro, 6GE pAECs showed significantly reduced binding of human IgM and IgG compared to wild-type pAECs (IgG p<0.01, IgM p<0.0001). Similar to TKO/hCD55 pAECs, 6GE pAECs exhibited a substantial reduction in complement-mediated cytotoxicity (p<0.001) compared to TKO pAECs. Co-expression of hTM and hEPCR in 6GE pigs led to a significant decrease in thrombin-antithrombin (TAT) complex levels in co-culture with human whole blood, compared to WT (p<0.0001), TKO (p<0.01), and TKO/hCD55/hTM pigs (p<0.05). Pathophysiological analysis demonstrated excellent compatibility of 6GE pig kidneys and livers with human immune and coagulation systems. However, 6GE pigs showed increased susceptibility to infection compared to other gene-edited pigs, while TKO/hCD55 pigs were considered safe when they were all bred in a general environment.
DISCUSSION: Highly expressing hCD55, along with the co-expression of hEPCR and hTM genes, is expected to effectively reduce human complement cytotoxicity and enhance anticoagulant efficacy in genetically modified pigs. The 6GE pigs exhibited robust compatibility with human physiological and immune systems, fulfilling the criteria for clinical trials. Furthermore, it is imperative to rear donor pigs in pathogen-free (DPF) facilities to mitigate infection risks and prevent the transmission of porcine pathogens to humans.}, }
@article {pmid39901848, year = {2025}, author = {Torigoe, N and Lin, Q and Liu, B and Nakayama, Y and Nakai, A and Nagahara, M and Tanihara, F and Hirata, M and Otoi, T}, title = {Effects of Electroporation Timing and Cumulus Cell Attachment on In Vitro Development and Genome Editing of Porcine Embryos.}, journal = {Reproduction in domestic animals = Zuchthygiene}, volume = {60}, number = {2}, pages = {e70011}, doi = {10.1111/rda.70011}, pmid = {39901848}, issn = {1439-0531}, support = {JP22H02499//Japan Society for the Promotion of Science/ ; JP22K19896//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; *Cumulus Cells ; *Electroporation/veterinary/methods ; *Gene Editing/methods/veterinary ; Female ; *Oocytes/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; Embryonic Development ; Swine ; Zygote ; RNA, Guide, CRISPR-Cas Systems ; Blastocyst/physiology ; Galactosyltransferases/genetics ; Embryo Culture Techniques/veterinary ; }, abstract = {Pig genome editing using the oviductal nucleic acid delivery (GONAD) method with electroporation would allow the efficient obtention of genetically modified pigs. However, oocytes and zygotes at various stages after ovulation must be targeted, and cumulus cell attachment and mosaic mutations are major obstacles. Therefore, we investigated whether two parameters (electroporation timing and the cumulus cell attachment) influence the effectiveness of multiplex genome editing by electroporation in porcine oocytes or zygotes. Three gRNAs targeting either GGTA1, CMAH or B4GALNT2 were introduced individually into oocytes and zygotes with and without cumulus cells at three different time points, 0 h before in vitro fertilisation (IVF) and 5 h and 10 h after IVF initiation. The introduction of gRNAs into oocytes and zygotes did not significantly affect the rates of blastocyst formation and total mutation of the resulting blastocysts irrespective of cumulus cell attachment and electroporation timing. In conclusion, the electroporation timing and the cumulus cell attachment did not interfere with the efficient delivery of the CRISPR/Cas9 system to the oocytes/zygotes, indicating that porcine genome editing in the oviduct using GONAD method may be possible.}, }
@article {pmid39901278, year = {2025}, author = {Zhang, D and Parth, F and da Silva, LM and Ha, TC and Schambach, A and Boch, J}, title = {Engineering a bacterial toxin deaminase from the DYW-family into a novel cytosine base editor for plants and mammalian cells.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {18}, pmid = {39901278}, issn = {1474-760X}, support = {BO 1496/9-1//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Gene Editing ; *Cytosine/metabolism ; *CRISPR-Cas Systems ; *Oryza/genetics ; Humans ; *Bacterial Toxins/genetics/metabolism ; Cytosine Deaminase/genetics/metabolism ; Animals ; Hordeum/genetics/enzymology ; Protoplasts/metabolism ; HEK293 Cells ; Mutation ; }, abstract = {Base editors are precise editing tools that employ deaminases to modify target DNA bases. The DYW-family of cytosine deaminases is structurally and phylogenetically distinct and might be harnessed for genome editing tools. We report a novel CRISPR/Cas9-cytosine base editor using SsdA, a DYW-like deaminase and bacterial toxin. A G103S mutation in SsdA enhances C-to-T editing efficiency while reducing its toxicity. Truncations result in an extraordinarily small enzyme. The SsdA-base editor efficiently converts C-to-T in rice and barley protoplasts and induces mutations in rice plants and mammalian cells. The engineered SsdA is a highly efficient genome editing tool.}, }
@article {pmid39899041, year = {2025}, author = {Lv, H and Sun, J and Guo, Y and Hang, G and Wu, Q and Sun, Z and Zhang, H}, title = {Isolation of Enterococcus hirae From Fresh White Yak Milk in Ledu District, Qinghai Province, China: A Comparative Genomic Analysis.}, journal = {Current microbiology}, volume = {82}, number = {3}, pages = {111}, pmid = {39899041}, issn = {1432-0991}, support = {U22A20540//Natural Science Foundation of China/ ; 2023KYPT0019//Inner Mongolia Science & Technology planning project/ ; BR22-12-03//Basic Research Operating Expenses Program for Colleges and Universities directly under the Inner Mongolia Autonomous Region/ ; }, mesh = {Animals ; *Milk/microbiology ; China ; *Enterococcus hirae/genetics/classification/isolation & purification ; Cattle ; *Genome, Bacterial ; Genomics ; Bacteriocins/genetics ; Virulence Factors/genetics ; Food Microbiology ; Phylogeny ; Humans ; }, abstract = {Yak milk is a widely consumed dairy product rich in lactic acid bacteria. Although Enterococcus hirae (E. hirae) is commonly found in dairy products and other foods, there is limited information available on its genetic makeup in yak milk. In the present study, 10 E. hirae strains isolated and identified from fresh white yak milk samples, along with 442 E. hirae strains obtained from the NCBI database (totaling 452 strains), were subjected to comparative genomic analysis. The findings of this study revealed that E. hirae has an open pan-genomic structure that allows for its high adaptability and environmental plasticity. Notably, E. hirae isolates from fresh white yak milk had smaller genomes, encoded more functional genes, and had fewer copies of genes encoding carbohydrate-active enzymes involved in the degradation of oligosaccharide metabolism and autolysin synthesis (CE1, GH73, GH23, and GT4 families) than those from animal and human isolates (P < 0.05). Additionally, fresh white yak milk isolates carried only three intrinsic bacteriocins and lacked virulence factors, CRISPR-Cas systems, and resistance genes linked to pathogenicity, which may be attributed to their specialization in the milk-derived environment. This study provides new insights into the genetic and functional gene diversity of E. hirae and how it adapts to milk-derived habitats.}, }
@article {pmid39899011, year = {2025}, author = {Kim, MB and Lee, YJ}, title = {Characterisation of the CRISPR-Cas systems in Enterococcus faecalis from commercial broiler farm environments and its association with antimicrobial resistance.}, journal = {British poultry science}, volume = {}, number = {}, pages = {1-8}, doi = {10.1080/00071668.2025.2451268}, pmid = {39899011}, issn = {1466-1799}, abstract = {1. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) systems have been highlighted for their potential applications in controlling the spread of mobile genetic elements, including antimicrobial resistance (AMR) genes. This study investigated the characteristics of CRISPR-Cas systems in E. faecalis from commercial broiler farms and assessed the impact of these systems on AMR.2. All E. faecalis isolates contained CRISPR2, and CRISPR1-Cas and CRISPR3-Cas were identified in 84 (56.4%) and 144 (96.6%) isolates. A combination of CRISPR2 and CRISPR3-Cas and a combination of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas were each identified in 27 (96.4%) farms.3. There were significant differences between CRISPR-Cas systems for phenotypic AMR: CRISPR1-Cas and CRISPR3-Cas. The E. faecalis isolates without CRISPR1-Cas showed higher resistance to most antimicrobials and had a higher prevalence of multidrug resistance (MDR) than those with CRISPR1-Cas. However, the resistance rate against most antimicrobials and the prevalence of MDR did not differ significantly depending on the presence or absence of CRISPR3-Cas.4. The E. faecalis isolates without CRISPR1-Cas harboured higher levels of all AMR genes, except for tetL, than those with CRISPR1-Cas. However, the E. faecalis isolates with CRISPR3-Cas showed a significant lower prevalence of tetL gene and a significantly higher prevalence of fexA and poxtA genes.5. In the distribution of rep families, the rep9 family was predominant, followed by rep1, rep7, rep2 and rep8 families. Only prevalence of the rep7 family was significantly higher in the E. faecalis isolates without CRISPR1-Cas (15.4%) than in those with CRISPR1-Cas (0%).6. This study is the first report on the characteristics of CRISPR-Cas systems in E. faecalis isolated from commercial broiler farm environments, and the results supported the hypothesis that the development of antimicrobial strategies requires an understanding of the distinctive capabilities between CRISPR1-Cas and CRISPR3-Cas and their underlying resistance mechanisms.}, }
@article {pmid39838618, year = {2025}, author = {Su, Y and Zhao, B and Zhang, L and Shen, B and Guo, C and Xiao, H and Li, N}, title = {Quantitative Analysis of Phosphorothioate Isomers in CRISPR sgRNA at Single-Residue Resolution Using Endonuclease Digestion Coupled with Liquid Chromatography Cyclic Ion Mobility Mass Spectrometry (LC/cIMS).}, journal = {Analytical chemistry}, volume = {97}, number = {4}, pages = {2223-2231}, doi = {10.1021/acs.analchem.4c05304}, pmid = {39838618}, issn = {1520-6882}, mesh = {*Mass Spectrometry/methods ; Phosphorothioate Oligonucleotides/chemistry/analysis ; Isomerism ; Ion Mobility Spectrometry/methods ; Chromatography, Liquid/methods ; Endonucleases/metabolism/chemistry ; CRISPR-Cas Systems/genetics ; Chromatography, Reverse-Phase/methods ; }, abstract = {Phosphorothioate (PS) modifications in single-guided RNA (sgRNA) are crucial for genome editing applications using the CRISPR/Cas9 system. These modifications may enhance sgRNA stability, pharmacokinetics, and binding to targets, thereby facilitating the desired genetic alterations. Incorporating multiple PS groups at varying positions may introduce chiral centers into the sgRNA backbone, resulting in a complex mixture of constitutional- and stereoisomers that challenges current analytical capabilities for reliable identification and quantification. In this study, we developed an innovative methodology that combines endonuclease digestion of sgRNA with ion pairing reversed-phase liquid chromatography coupled with cyclic ion mobility mass spectrometry (IPRP-LC/cIMS) to fully distinguish PS-induced isomers in a complex mixture. The relative abundance of each isomer was quantified using a two-step method, wherein the ion abundance was sequentially extracted from an LC/MS ion chromatogram and LC/cIMS two-dimensional ion mobiligram. This quantification method was thoroughly evaluated, demonstrating excellent sensitivity, precision, dynamic range, repeatability, and accuracy. In addition, this method enables the investigation of the kinetics of forming PS to phosphodiester (PO) impurities in sgRNA under oxidative stress conditions, offering unprecedented insights into PS stability at a single-residue resolution. In this context, this method highlights the in-depth characterization of PS, demonstrating its capability to support biomedical research, development, and production of sgRNA products.}, }
@article {pmid39829104, year = {2025}, author = {Yang, L and Zhang, J and Zhang, J and Hou, T and Gao, Q and Liu, X and Li, F}, title = {CRISPR-Based Homogeneous Electrochemical Strategy for Near-Zero Background Detection of Breast Cancer Extracellular Vesicles via Fluidity-Enhanced Magnetic Capture Nanoprobe.}, journal = {Analytical chemistry}, volume = {97}, number = {4}, pages = {2176-2185}, doi = {10.1021/acs.analchem.4c05181}, pmid = {39829104}, issn = {1520-6882}, mesh = {Humans ; *Breast Neoplasms/diagnosis ; *Electrochemical Techniques ; *Extracellular Vesicles/chemistry ; Female ; Magnetite Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; }, abstract = {Precise identification and analysis of multiple protein biomarkers on the surface of breast cancer cell-derived extracellular vesicles (BC-EVs) are of great significance for noninvasive diagnosis of the breast cancer subtypes, but it remains a major challenge owing to their high heterogeneity and low abundance. Herein, we established a CRISPR-based homogeneous electrochemical strategy for near-zero background and ultrasensitive detection of BC-EVs. To realize the high-performance capture and isolation of BC-EVs, fluidity-enhanced magnetic nanoprobes were facilely prepared. After capturing BC-EVs, the AND logic gate-based catalytic hairpin assembly (CHA) and the trans-cleavage activity of CRISPR-Cas12a against the magnetic signal nanoprobes were triggered successively, generating a significant electrochemical signal. Notably, the as-developed metal-mediated magnetic signal nanoprobes could efficiently decrease the background signal by magnetic separation, endowing the method with a high signal-to-noise ratio. Consequently, by ingeniously integrating DNA logic gate-based CRISPR-CHA signal amplification with dual magnetic nanoprobes in a homogeneous electrochemical strategy, precise identification and ultrasensitive detection of BC-EVs was successfully achieved through simultaneous and specific recognition of dual protein markers on the BC-EVs surface. More importantly, this approach could effectively discriminate specific subgroups of BC-EVs in clinical serum samples, which may provide great opportunities for the accurate diagnosis and prognosis evaluation of breast cancer in a noninvasive manner.}, }
@article {pmid39817779, year = {2025}, author = {Wiull, K and Haugen, LK and Eijsink, VGH and Mathiesen, G}, title = {CRISPR/Cas9-mediated genomic insertion of functional genes into Lactiplantibacillus plantarum WCFS1.}, journal = {Microbiology spectrum}, volume = {13}, number = {2}, pages = {e0202524}, doi = {10.1128/spectrum.02025-24}, pmid = {39817779}, issn = {2165-0497}, support = {//Norges Miljø- og Biovitenskapelige Universitet (NMBU)/ ; }, mesh = {*CRISPR-Cas Systems ; *Plasmids/genetics ; *Gene Editing/methods ; Lactobacillus plantarum/genetics ; SARS-CoV-2/genetics ; Gene Knock-In Techniques/methods ; Mutagenesis, Insertional ; Humans ; }, abstract = {UNLABELLED: Lactiplantibacillus plantarum, a natural inhabitant of the human body, is a promising candidate vehicle for vaccine delivery. An obstacle in developing bacterial delivery vehicles is generating a production strain that lacks antibiotic resistance genes and contains minimal foreign DNA. To deal with this obstacle, we have constructed a finetuned, inducible two-plasmid CRISPR/Cas9-system for chromosomal gene insertion in L. plantarum. The knock-in plasmid was designed with a cassette-like structure to simplify the insertion of target DNA and streamline the CRISPR/Cas9 genome editing, bringing it one step closer to becoming a routine procedure. We demonstrate that the system enables efficient insertion of expression cassettes for both inducible and constitutive production of a fluorescent reporter protein, mCherry, and for inducible production of the receptor-binding domain (RBD) of the SARS-CoV-2 virus. Two variants of RBD were successfully expressed, one directed to the cytoplasm and one directed to the cell surface. All the knock-in strains produced the target protein, although with lower yields than strains with plasmid-encoded expression.
IMPORTANCE: Genetic engineering of lactic acid bacteria, such as Lactiplantibacillus plantarum, has proven to be difficult. This study presents an inducible two-plasmid CRISPR/Cas9-system for inserting genes into the chromosome of Lactiplantibacillus plantarum. Our system successfully knock-in four expression cassettes varying in length from ~800-1,300 bp with high efficiency and insert an expression cassette encoding a SARS-CoV-2 antigen receptor-binding domain (RBD) with an anchor mediating surface display, which has not been achieved previously using CRISPR/Cas9. We demonstrate the production of the insertion genes. Importantly, the plasmid carrying the SgRNA, Cas9, and homology-directed repair template is designed for easy component exchange. These plasmids represent valuable contributions to the field as they could facilitate rapid CRISPR/Cas9 engineering of L. plantarum strains.}, }
@article {pmid39776154, year = {2025}, author = {Carvalho, JE and Burtin, M and Detournay, O and Amiel, AR and Röttinger, E}, title = {Optimized husbandry and targeted gene-editing for the cnidarian Nematostella vectensis.}, journal = {Development (Cambridge, England)}, volume = {152}, number = {2}, pages = {}, doi = {10.1242/dev.204387}, pmid = {39776154}, issn = {1477-9129}, support = {ANR-15-IDEX-01//Agence Nationale de la Recherche/ ; ANR-20-CE13-014//Agence Nationale de la Recherche/ ; SPF20170938703//Fondation pour la Recherche Médicale/ ; SPF20130526781//Fondation pour la Recherche Médicale/ ; N/A//Région SUD/ ; DBM//Institut des sciences biologiques/ ; //Centre national de la Recherche Scientifique/ ; }, mesh = {Animals ; *Sea Anemones/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Aquaculture/methods ; Animal Husbandry/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Transgenes ; Animals, Genetically Modified ; }, abstract = {Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes controlling the entire life cycle, having access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model, to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies, juvenile growth and survival rates, and considerably reduced the overall life cycle down to 2 months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full knockout animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In summary, our study provides additional resources for the scientific community that uses or plans to use N. vectensis as a research model.}, }
@article {pmid39898483, year = {2025}, author = {Guerra-Resendez, RS and Lydon, SL and Ma, AJ and Bedford, GC and Reed, DR and Kim, S and Terán, ER and Nishiguchi, T and Escobar, M and DiNardo, AR and Hilton, IB}, title = {Characterization of Rationally Designed CRISPR/Cas9-Based DNA Methyltransferases with Distinct Methyltransferase and Gene Silencing Activities in Human Cell Lines and Primary Human T Cells.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00569}, pmid = {39898483}, issn = {2161-5063}, abstract = {Nuclease-deactivated Cas (dCas) proteins can be used to recruit epigenetic effectors, and this class of epigenetic editing technologies has revolutionized the ability to synthetically control the mammalian epigenome and transcriptome. DNA methylation is one of the most important and well-characterized epigenetic modifications in mammals, and while many different forms of dCas-based DNA methyltransferases (dCas-DNMTs) have been developed for programmable DNA methylation, these tools are frequently poorly tolerated and/or lowly expressed in mammalian cell types. Further, the use of dCas-DNMTs has largely been restricted to cell lines, which limits mechanistic insights in karyotypically normal contexts and hampers translational utility in the longer term. Here, we extend previous insights into the rational design of the catalytic core of the mammalian DNMT3A methyltransferase and test three dCas9-DNMT3A/3L variants across different human cell lines and in primary donor-derived human T cells. We find that mutations within the catalytic core of DNMT3A stabilize the expression of dCas9-DNMT3A/3L fusion proteins in Jurkat T cells without sacrificing DNA methylation or gene-silencing performance. We also show that these rationally engineered mutations in DNMT3A alter DNA methylation profiles at loci targeted with dCas9-DNMT3A/3L in cell lines and donor-derived human T cells. Finally, we leverage the transcriptionally repressive effects of dCas9-DNMT3A/3L variants to functionally link the expression of a key immunomodulatory transcription factor to cytokine secretion in donor-derived T cells. Overall, our work expands the synthetic biology toolkit for epigenetic editing and provides a roadmap for the use of engineered dCas-based DNMTs in primary mammalian cell types.}, }
@article {pmid39895071, year = {2025}, author = {Choi, YJ and Eom, H and Nandre, R and Kim, M and Oh, YL and Kim, S and Ro, HS}, title = {Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein.}, journal = {Journal of microbiology (Seoul, Korea)}, volume = {63}, number = {1}, pages = {e.2409006}, doi = {10.71150/jm.2409006}, pmid = {39895071}, issn = {1976-3794}, support = {2023R1A 2C1007213//National Research Foundation of Korea/ ; //Rural Development Administration/ ; RS-2024-00322425//New Breeding Technologies Development Program/ ; }, mesh = {*Gene Editing/methods ; *Cell Nucleus/metabolism/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Reishi/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; Orotidine-5'-Phosphate Decarboxylase/genetics/metabolism ; Protoplasts/metabolism ; }, abstract = {The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.}, }
@article {pmid39894896, year = {2025}, author = {Banday, S and Mishra, AK and Rashid, R and Ye, T and Ali, A and Li, J and Yustein, JT and Kelliher, MA and Zhu, LJ and Deibler, SK and Malonia, SK and Green, MR}, title = {The O-glycosyltransferase C1GALT1 promotes EWSR1::FLI1 expression and is a therapeutic target for Ewing sarcoma.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1267}, pmid = {39894896}, issn = {2041-1723}, mesh = {Humans ; Animals ; *Galactosyltransferases/genetics/metabolism ; *RNA-Binding Protein EWS/genetics/metabolism ; Cell Line, Tumor ; *Sarcoma, Ewing/genetics/metabolism/pathology/drug therapy ; Mice ; *Oncogene Proteins, Fusion/genetics/metabolism ; *Gene Expression Regulation, Neoplastic ; Proto-Oncogene Protein c-fli-1/metabolism/genetics ; Bone Neoplasms/genetics/pathology/metabolism/drug therapy ; Signal Transduction/drug effects ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems/genetics ; Hedgehog Proteins/metabolism/genetics ; Female ; }, abstract = {Ewing sarcoma (ES) is an aggressive bone cancer driven by the oncogenic fusion-protein EWSR1::FLI1, which is not present in normal cells and is therefore an attractive therapeutic target. However, as a transcription factor, EWSR1::FLI1 is considered undruggable. Factors that promote EWSR1::FLI1 expression, and thus whose inhibition would reduce EWSR1::FLI1 protein levels and function, are potential drug targets. Here, using genome-scale CRISPR/Cas9 knockout screening, we identify C1GALT1, a galactosyltransferase required for the biosynthesis of many O-glycoproteins, as a factor that promotes EWSR1::FLI1 expression. We show that C1GALT1 acts by O-glycosylating the pivotal Hedgehog (Hh) signaling component Smoothened (SMO), thereby stabilizing SMO and stimulating the Hh pathway, which we find directly activates EWSR1::FLI1 transcription. Itraconazole, an FDA-approved anti-fungal agent that is known to inhibit C1GALT1, reduces EWSR1::FLI1 levels in ES cell lines and suppresses growth of ES xenografts in mice. Our study reveals a therapeutically targetable mechanism that promotes EWSR1::FLI1 expression and ES tumor growth.}, }
@article {pmid39894193, year = {2025}, author = {Saini, A and Dilbaghi, N and Yadav, N}, title = {CRISPR integrated biosensors: A new paradigm for cancer detection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {569}, number = {}, pages = {120179}, doi = {10.1016/j.cca.2025.120179}, pmid = {39894193}, issn = {1873-3492}, abstract = {Cancer remains one of the leading causes of morbidity and mortality globally, necessitating need for advancements of technologies for early therapeutics. Conventional detection methodologies often lag behind in terms of sensitivity, specificity, and cost-effectiveness, leading to delayed diagnosis and inadequate treatment. The need of advanced diagnostic techniques has considerably increased and led to the development of biosensors. Biosensing technologies offer several advantages over conventional methods hence, overcome limitations and improve diagnostic accuracy. Biosensors, particularly CRISPR-Cas based biosensors have emerged as a revolutionary technology for oncology diagnostics due to their high precision and adaptability. CRISPR-based biosensors provide remarkable precision, sensitivity, multiplexing capabilities, specificity, and rapidness for developing a cost-effective and portable point of care diagnostic device for cancer detection. In this review, we have discussed cancer pathogenicity, assessed the traditional detection techniques, and explored the advancements and advantages of biosensors, particularly CRISPR-based biosensors, in the detection of some major cancer types, namely lung, liver, colorectal, prostate, and cervical cancers. CRISPR-based biosensors represent a significant potential in cancer diagnostics, offering precise, cost-effective, and rapid detection of cancer biomarkers. The integration of CRISPR technology with biosensors holds substantial promise for enhancing early detection and improving patient outcomes in cancer diagnostics.}, }
@article {pmid39841710, year = {2025}, author = {Park, BJ and Heo, ST and Kim, M and Yoo, JR and Bae, EJ and Kang, SY and Park, S and Han, KR and Lee, KH and Lee, JM and Lee, H and Song, YJ}, title = {A CRISPR-Cas12a-based universal rapid scrub typhus diagnostic method targeting 16S rRNA of Orientia tsutsugamushi.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012826}, doi = {10.1371/journal.pntd.0012826}, pmid = {39841710}, issn = {1935-2735}, mesh = {*Scrub Typhus/diagnosis/microbiology ; *Orientia tsutsugamushi/genetics/isolation & purification ; *RNA, Ribosomal, 16S/genetics ; Humans ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Molecular Diagnostic Techniques/methods ; Polymerase Chain Reaction/methods ; }, abstract = {Scrub typhus is caused by Orientia tsutsugamushi infection and occurs frequently in an area called the Tsutsugamushi Triangle. Currently, there is no vaccine for O. tsutsugamushi, and its infection is treated with antibiotics such as doxycycline. Scrub typhus responds to effective treatment, and early treatment shortens the course of the disease, reduces mortality, and accelerates recovery. Therefore, it is important to rapidly diagnose O. tsutsugamushi infection to ensure successful outcomes. Here, we developed a CRISPR-Cas12a-based diagnostic method targeting the bacterial 16S rRNA to detect O. tsutsugamushi infection of all known genotypes. To reduce the possibility of contamination and increase field applicability, we designed the one-pot assay system in addition to conventional two-pot assay system. Using this method, we successfully detected up to 100 copies of in vitro transcribed O. tsutsugamushi 16S rRNA within 1 hour under isothermal conditions. In blood samples from patients confirmed to be infected with O. tsutsugamushi by nested PCR, the developed method exhibited a clinical sensitivity of 98% and high specificity. These data demonstrate that the presented method is applicable for the rapid and universal diagnosis of scrub typhus to facilitate timely and appropriate treatment.}, }
@article {pmid39817463, year = {2025}, author = {Zuo, D and Liu, W and Zhao, R and Zhu, K and Wang, W and Xiang, H}, title = {Drosophila CG11700 may not affect male fecundity-lifespan tradeoff as previously reported.}, journal = {Molecular biology and evolution}, volume = {42}, number = {2}, pages = {}, doi = {10.1093/molbev/msaf003}, pmid = {39817463}, issn = {1537-1719}, mesh = {Animals ; Male ; *Fertility/genetics ; *Longevity/genetics ; *Drosophila Proteins/genetics ; CRISPR-Cas Systems ; Female ; Drosophila/genetics ; Drosophila melanogaster/genetics ; }, abstract = {Our recent investigations on the function of Drosophila CG11700 and CG32744 (Ubi-p5E) genes using CRISPR/Cas9 deletion technology could not repeat or confirm the results on CG11700 shown in our previous study, which was based on P-element excision assay (Zhan Z, Ding Y, Zhao R, Zhang Y, Yu H, Zhou Q, Yang S, Xiang H, Wang W. Rapid functional divergence of a newly evolved polyubiquitin gene in Drosophila and its role in the trade-off between male fecundity and lifespan. Mol Biol Evol. 2012:29(5):1407-1416. doi:10.1093/molbev/msr299). Here, by CRISPR/Cas9 editing, we generated mutants of CG32744 with the whole gene body fully deleted from the genome, and truncated mutants of CG11700 with N-terminal 103 aa deleted out of its total 301 aa peptide sequence. We carefully conducted the male fecundity assay and found that offsprings of the CG11700 mutant were not significantly more than the wild type, inconsistent with our previous report (Zhan et al. 2012). Meanwhile, we repeated the lifespan assay and did not find that the lifespan of the CG11700 mutant was significantly shorter than the wild type as reported (2012). The new results suggest that the CG11700 gene may not affect male fecundity-lifespan tradeoff as previously reported (Zhan et al. 2012). The new results are thus worthy of reporting to avoid possible misleading by the previous results to the scientific community.}, }
@article {pmid39671912, year = {2025}, author = {Zhao, D and Deshpande, R and Wu, K and Tyagi, A and Sharma, S and Wu, SY and Xing, F and O'Neill, S and Ruiz, J and Lyu, F and Watabe, K}, title = {Identification of TUBB3 as an immunotherapy target in lung cancer by genome wide in vivo CRISPR screening.}, journal = {Neoplasia (New York, N.Y.)}, volume = {60}, number = {}, pages = {101100}, pmid = {39671912}, issn = {1476-5586}, support = {P30 CA012197/CA/NCI NIH HHS/United States ; R01 CA173499/CA/NCI NIH HHS/United States ; R01 CA185650/CA/NCI NIH HHS/United States ; R01 CA205067/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Lung Neoplasms/genetics/immunology/drug therapy/therapy/pathology ; *Tubulin/genetics/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Xenograft Model Antitumor Assays ; Drug Resistance, Neoplasm/genetics ; T-Lymphocytes, Cytotoxic/immunology ; Disease Models, Animal ; Snail Family Transcription Factors/genetics/metabolism ; }, abstract = {Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer.}, }
@article {pmid39668248, year = {2025}, author = {Philip, R and Sharma, A and Matellan, L and Erpf, AC and Hsu, WH and Tkach, JM and Wyatt, HDM and Pelletier, L}, title = {qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.}, journal = {The EMBO journal}, volume = {44}, number = {3}, pages = {947-974}, pmid = {39668248}, issn = {1460-2075}, support = {187836//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 181763//Canadian Government | CIHR | Institute of Cancer Research (IC)/ ; 156297//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; 167279//Canadian Government | Canadian Institutes of Health Research (CIHR)/ ; N/A//Krembil Foundation/ ; }, mesh = {*Plasmids/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.}, }
@article {pmid39893465, year = {2025}, author = {Li, B and Shang, Y and Wang, L and Lv, J and Wu, Q and Wang, F and Chao, J and Mao, J and Ding, A and Wu, X and Xue, K and Chen, C and Cui, M and Sun, Y and Zhang, H and Dai, C}, title = {Efficient genome editing in dicot plants using calreticulin promoter-driven CRISPR/Cas system.}, journal = {Molecular horticulture}, volume = {5}, number = {1}, pages = {9}, pmid = {39893465}, issn = {2730-9401}, }
@article {pmid39893181, year = {2025}, author = {Hu, L and Han, J and Wang, HD and Cheng, ZH and Lv, CC and Liu, DF and Yu, HQ}, title = {A universal and wide-range cytosine base editor via domain-inlaid and fidelity-optimized CRISPR-FrCas9.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1260}, pmid = {39893181}, issn = {2041-1723}, support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {*Cytosine/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Escherichia coli/genetics/metabolism ; Pseudomonas aeruginosa/genetics ; Protein Domains ; Deoxyribonuclease I/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mutation ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {CRISPR-based base editor (BE) offer diverse editing options for genetic engineering of microorganisms, but its application is limited by protospacer adjacent motif (PAM) sequences, context preference, editing window, and off-target effects. Here, a series of iteratively improved cytosine base editors (CBEs) are constructed using the FrCas9 nickase (FrCas9n) with the unique PAM palindromic structure (NNTA) to alleviate these challenges. The deaminase domain-inlaid FrCas9n exhibits an editing range covering 38 nucleotides upstream and downstream of the palindromic PAM, without context preference, which is 6.3 times larger than that of traditional CBEs. Additionally, lower off-target editing is achieved when incorporating high-fidelity mutations at R61A and Q964A in FrCas9n, while maintaining high editing efficiency. The final CBE, HF-ID824-evoCDA-FrCas9n demonstrates broad applicability across different microbes such as Escherichia coli MG1655, Shewanella oneidensis MR-1, and Pseudomonas aeruginosa PAO1. Collectively, this tool offers robust gene editing for facilitating mechanistic studies, functional exploration, and protein evolution in microbes.}, }
@article {pmid39893176, year = {2025}, author = {Sulis, DB and Lavoine, N and Sederoff, H and Jiang, X and Marques, BM and Lan, K and Cofre-Vega, C and Barrangou, R and Wang, JP}, title = {Advances in lignocellulosic feedstocks for bioenergy and bioproducts.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1244}, pmid = {39893176}, issn = {2041-1723}, mesh = {*Lignin/metabolism ; *Biofuels ; *Gene Editing ; Biotechnology/methods ; Biomass ; CRISPR-Cas Systems ; }, abstract = {Lignocellulose, an abundant renewable resource, presents a promising alternative for sustainable energy and industrial applications. However, large-scale adoption of lignocellulosic feedstocks faces considerable obstacles, including scalability, bioprocessing efficiency, and resilience to climate change. This Review examines current efforts and future opportunities for leveraging lignocellulosic feedstocks in bio-based energy and products, with a focus on enhancing conversion efficiency and scalability. It also explores emerging biotechnologies such as CRISPR-based genome editing informed by machine learning, aimed at improving feedstock traits and reducing the environmental impact of fossil fuel dependence.}, }
@article {pmid39891928, year = {2025}, author = {Madorsky Rowdo, FP and Martini, R and Ackermann, SE and Tang, CP and Tranquille, M and Irizarry, A and Us, I and Alawa, O and Moyer, JE and Sigouros, M and Nguyen, J and Al Assaad, M and Cheng, E and Ginter, PS and Manohar, J and Stonaker, B and Boateng, R and Oppong, JK and Adjei, EK and Awuah, B and Kyei, I and Aitpillah, FS and Adinku, MO and Ankomah, K and Osei-Bonsu, EB and Gyan, KK and Hoda, S and Newman, L and Mosquera, JM and Sboner, A and Elemento, O and Dow, LE and Davis, MB and Martin, ML}, title = {Kinome-Focused CRISPR-Cas9 Screens in African Ancestry Patient-Derived Breast Cancer Organoids Identify Essential Kinases and Synergy of EGFR and FGFR1 Inhibition.}, journal = {Cancer research}, volume = {85}, number = {3}, pages = {551-566}, doi = {10.1158/0008-5472.CAN-24-0775}, pmid = {39891928}, issn = {1538-7445}, support = {//Weill Cornell Medicine (WCM)/ ; BCRF-22-191//Breast Cancer Research Foundation (BCRF)/ ; CA259396-01//National Cancer Institute (NCI)/ ; }, mesh = {Humans ; *Organoids/pathology/metabolism ; *Breast Neoplasms/genetics/pathology/drug therapy ; Female ; *CRISPR-Cas Systems ; *Receptor, Fibroblast Growth Factor, Type 1/genetics/antagonists & inhibitors/metabolism ; *ErbB Receptors/genetics/antagonists & inhibitors/metabolism ; Protein Kinase Inhibitors/pharmacology ; Black People/genetics ; }, abstract = {Precision medicine approaches to cancer treatment aim to exploit genomic alterations that are specific to individual patients to tailor therapeutic strategies. Yet, some targetable genes and pathways are essential for tumor cell viability even in the absence of direct genomic alterations. In underrepresented populations, the mutational landscape and determinants of response to existing therapies are poorly characterized because of limited inclusion in clinical trials and studies. One way to reveal tumor essential genes is with genetic screens. Most screens are conducted on cell lines that bear little resemblance to patient tumors, after years of culture under nonphysiologic conditions. To address this problem, we aimed to develop a CRISPR screening pipeline in three-dimensionally grown patient-derived tumor organoid (PDTO) models. A breast cancer PDTO biobank that focused on underrepresented populations, including West African patients, was established and used to conduct a negative-selection kinome-focused CRISPR screen to identify kinases essential for organoid growth and potential targets for combination therapy with EGFR or MEK inhibitors. The screen identified several previously unidentified kinase targets, and the combination of FGFR1 and EGFR inhibitors synergized to block organoid proliferation. Together, these data demonstrate the feasibility of CRISPR-based genetic screens in patient-derived tumor models, including PDTOs from underrepresented patients with cancer, and identify targets for cancer therapy. Significance: Generation of a breast cancer patient-derived tumor organoid biobank focused on underrepresented populations enabled kinome-focused CRISPR screening that identified essential kinases and potential targets for combination therapy with EGFR or MEK inhibitors. See related commentary by Trembath and Spanheimer, p. 407.}, }
@article {pmid39891927, year = {2025}, author = {Trembath, HE and Spanheimer, PM}, title = {In Search of Representative Translational Cancer Model Systems.}, journal = {Cancer research}, volume = {85}, number = {3}, pages = {407-409}, doi = {10.1158/0008-5472.CAN-24-3879}, pmid = {39891927}, issn = {1538-7445}, mesh = {Humans ; *Organoids/pathology ; Female ; Breast Neoplasms/genetics/pathology/therapy ; CRISPR-Cas Systems ; Precision Medicine/methods ; Translational Research, Biomedical/methods ; Neoplasms/genetics/pathology/therapy ; Tumor Microenvironment ; }, abstract = {Racial disparities in cancer outcomes are well documented across tumor types. For patients with breast cancer, Black women are more likely to present with more aggressive molecular features and more likely to die from disease, even after accounting for those features. Recent efforts have been aimed at developing translational model systems for precision medicine strategies, and a major focus has been on patient-derived organoids. Organoids allow for robust in vitro experimental platforms, including drug and CRISPR screens while maintaining more complex cancer and tumor microenvironment subpopulations than cell lines. For results that are broadly translationally relevant, it is important that cancer models are derived from the spectrum of human disease and humans with disease. In this issue of Cancer Research, Madorsky Rowdo and colleagues derive breast cancer organoids from patients with African ancestry and use CRISPR-Cas9 screens to identify novel therapeutic vulnerabilities. These findings demonstrate the promise of representative cancer model systems to facilitate discoveries that are most likely to translate to improved therapy for all patients. See related article by Madorsky Rowdo et al., p. 551.}, }
@article {pmid39891757, year = {2025}, author = {Roychowdhury, R and Das, SP and Das, S and Biswas, S and Patel, MK and Kumar, A and Sarker, U and Choudhary, SP and Das, R and Yogendra, K and Gangurde, SS}, title = {Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.}, journal = {Functional & integrative genomics}, volume = {25}, number = {1}, pages = {31}, pmid = {39891757}, issn = {1438-7948}, mesh = {*Gene Editing ; *Food Security ; *Climate Change ; *Vegetables/metabolism/genetics ; *CRISPR-Cas Systems ; Plant Breeding/methods ; Crops, Agricultural/genetics ; Humans ; }, abstract = {As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances.}, }
@article {pmid39890862, year = {2025}, author = {Nalefski, EA and Sinan, S and Cantera, JL and Kim, AG and Kooistra, RM and Rivera, RE and Janshen, JP and Bhadra, S and Bishop, JD and Ellington, AD and Finklestein, IJ and Madan, D}, title = {Room temperature CRISPR diagnostics for low-resource settings.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {3909}, pmid = {39890862}, issn = {2045-2322}, mesh = {Humans ; *CRISPR-Cas Systems ; *Temperature ; Endodeoxyribonucleases/genetics/metabolism ; Human papillomavirus 16/genetics/isolation & purification ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins/genetics ; Female ; Recombinases/metabolism ; Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; Sensitivity and Specificity ; }, abstract = {Maintaining elevated reaction temperatures and multi-step sample preparations increases the costs and complexity of diagnostics, impeding their deployment in low-resource settings. Here, we develop a one-pot, room temperature recombinase polymerase amplification (RPA)-CRISPR reaction that removes these critical challenges. We show that RPA amplification is reduced by several orders of magnitude at 25 °C as compared to 37 °C. Similarly, when coupled to RPA, the performance of multiple Cas12a orthologs, including the widely used LbCas12a, is severely compromised at temperatures below 37 °C. To mitigate these limitations, we identify the ortholog TsCas12a as a highly active nuclease at 25 °C and develop a single-protocol RPA-Cas12a detection reaction with this enzyme. A quantitative kinetic analysis reveals that fast nuclease activation is more critical than higher steady-state trans-cleavage activity for room temperature diagnostic applications. RPA-TsCas12a reactions performed at 25 °C effectively detected HPV-16 in crudely prepared cervical swab samples with high sensitivity and specificity using both optical and lateral flow readouts. The reactions developed herein reduce the complexity and equipment requirements for affordable diagnostics in low- and middle-income countries.}, }
@article {pmid39862677, year = {2025}, author = {Pei, J and Li, L and Li, C and Li, Z and Wu, Y and Kuang, H and Ma, P and Huang, L and Liu, J and Tian, G}, title = {Dumbbell probe-bridged CRISPR/Cas13a and nicking-mediated DNA cascade reaction for highly sensitive detection of colorectal cancer-related microRNAs.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117190}, doi = {10.1016/j.bios.2025.117190}, pmid = {39862677}, issn = {1873-4235}, mesh = {*Colorectal Neoplasms/diagnosis/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; DNA/chemistry/genetics ; Electrochemical Techniques/methods ; }, abstract = {Colorectal cancer (CRC) is a leading cause of cancer-related deaths globally, necessitating the development of sensitive and minimally invasive diagnostic approaches. In this study, we present a novel diagnostic strategy by integrating dumbbell probe-mediated CRISPR/Cas13a with nicking-induced DNA cascade reaction (DP-bridged Cas13a/NDCR) for highly sensitive microRNA (miRNA) detection. Target miRNA triggers Cas13a-mediated cleavage of the dumbbell probe, releasing an intermediate strand that hybridizes with a methylene blue-labeled hairpin probe on the electrode surface. Nicking enzyme cleaves the formed duplex DNA, triggering a cascade reaction that amplifies the electrochemical signal. Under optimized conditions, the method demonstrates a detection limit of 8.26 fM for miRNA-21, with reliable specificity and long-term stability. Furthermore, integration with machine learning models using multiple miRNA markers improved diagnostic accuracy, differentiating CRC from colorectal polyps and healthy controls with 100% accuracy in clinical validation cohorts. This study highlights the potential of DP-bridged Cas13a/NDCR as a sensitive and accurate diagnostic tool for CRC.}, }
@article {pmid39842078, year = {2025}, author = {Kumar, ARK and Low, J and Lim, J and Myint, B and Sun, X and Wu, L and Cheng, HS and Yip, S and Ming Cheng, CZ and Manoharan, T and Quek, YJ and Shou, Y and Tian, JS and Ng, YY and Gascoigne, NRJ and Tan, NS and Sugimura, R and Chia, G and Sze Cheung, AM and Yawata, M and Tay, A}, title = {Non-viral, high throughput genetic engineering of primary immune cells using nanostraw-mediated transfection.}, journal = {Biomaterials}, volume = {317}, number = {}, pages = {123079}, doi = {10.1016/j.biomaterials.2024.123079}, pmid = {39842078}, issn = {1878-5905}, mesh = {Humans ; *Transfection/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; CD8-Positive T-Lymphocytes/immunology ; Electroporation/methods ; CD4-Positive T-Lymphocytes/immunology ; }, abstract = {Transfection of proteins, mRNA, and chimeric antigen receptor (CAR) transgenes into immune cells remains a critical bottleneck in cell manufacturing. Current methods, such as viruses and bulk electroporation, are hampered by low transfection efficiency, unintended transgene integration, and significant cell perturbation. The Nanostraw Electro-actuated Transfection (NExT) technology offers a solution by using high aspect-ratio nanostraws and localized electric fields to precisely deliver biomolecules into cells with minimal disruption. We demonstrate that NExT can deliver proteins, polysaccharides, and mRNA into primary human CD8[+] and CD4[+] T cells, and achieve CRISPR/Cas9 gene knockout of CXCR4 and TRAC in CD8[+] T cells. We showcase NExT's versatility across a range of primary human immune cells, including CD4[+] T cells, γδ-T cells, dendritic cells, NK cells, Treg cells, macrophages, and neutrophils. Finally, we developed a scalable, high-throughput multiwell NExT system capable of transfecting over 14 million cells and delivering diverse cargoes into multiple cell types from various donors simultaneously. This technology holds promise for streamlining high-throughput screening of allogeneic donors and reducing optimization costs for large-scale CAR-immune cell transfection.}, }
@article {pmid39842058, year = {2025}, author = {Jia, HY and Yao, SY and Li, YF and Ye, BC and Yin, BC}, title = {A conformational switch-controlled RNA sensor based on orthogonal dCas12a for RNA imaging in live cells.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117185}, doi = {10.1016/j.bios.2025.117185}, pmid = {39842058}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; Humans ; *RNA/chemistry/genetics ; *CRISPR-Cas Systems ; CRISPR-Associated Proteins/chemistry ; Bacterial Proteins/chemistry/genetics ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry ; Fluorescent Dyes/chemistry ; }, abstract = {RNA imaging technology is essential for understanding the complex RNA regulatory mechanisms and serves as a powerful tool for disease diagnosis. However, conventional RNA imaging methods often require multiple fluorescent tags for the specific labeling of individual targets, complicating both the imaging process and subsequent analysis. Herein, we develop an RNA sensor that integrates a blocked CRISPR RNA (crRNA)-based conformational switch with a controllable CRISPR activation (CRISPRa) system and apply for RNA imaging. By leveraging nuclease-inactive Cas12a (dCas12a)-mediated processing of precursor crRNA (pre-crRNA) and the orthogonality of dCas12a from different bacteria, our sensor establishes an artificial link between two unrelated RNA targets, enabling cells to sense one RNA target and image another with a single fluorescent signal. By visualizing a single target for dual-target analysis, our method significantly reduces the reliance on multiple fluorescent tags. Our sensor provides a new platform for RNA imaging, enhancing both biomedical research and the development of advanced molecular diagnostics.}, }
@article {pmid39837235, year = {2025}, author = {Ding, S and Dong, J and Shi, J and Ren, K and Cui, X and Shi, Z and Li, N and Xiang, Y and Du, F and Tang, Z}, title = {Integrating commercial personal glucose meter with peroxidase-mimic DNAzyme to develop a versatile point-of-care biosensing platform.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117171}, doi = {10.1016/j.bios.2025.117171}, pmid = {39837235}, issn = {1873-4235}, mesh = {*DNA, Catalytic/chemistry ; *Biosensing Techniques/instrumentation/methods ; Humans ; *SARS-CoV-2/isolation & purification ; *COVID-19/diagnosis/virology ; *Blood Glucose Self-Monitoring/instrumentation ; Point-of-Care Testing ; Point-of-Care Systems ; Peroxidase/chemistry ; Glucose/analysis ; Blood Glucose/analysis ; CRISPR-Cas Systems ; RNA, Viral/analysis ; Limit of Detection ; }, abstract = {The development of point-of-care testing (POCT) methods is highly desirable in molecular detection, as they enable disease diagnosis and biomarker monitoring on-site or at home. Repurposing existing POCT devices to detect diverse biomarkers is an economical way to develop new devices for POCT use. Personal glucose meter (PGM) is one of the most used off-the-shelf POCT devices that has been reused to detect non-glucose targets. However, developing a label-free, user-friendly, and cost-effective general PGM-based sensing platform remains a great challenge, primarily due to the reliance on protein enzymes in most existing signal transducing strategies. To overcome the challenges, we herein developed a DNAzyme-based signal transduction strategy that bridges non-glucose signals to PGM readouts. By integrating this strategy with CRISPR/Cas12a-mediated target sensing, we successfully established a simple and versatile platform (CaG-PGM) for biosensing. The utility of CaG-PGM in the detection of nucleic acid targets was successfully validated by detecting Monkeypox virus DNA and SARS-CoV-2 RNA with high sensitivity and specificity. We further demonstrated its generality in detecting non-nucleic acid targets including protein and small molecule. In conclusion, this study provides a cheap and effective strategy for repurposing PGM as a general biosensing platform and sheds new light on translating functional nucleic acids for POCT applications.}, }
@article {pmid39832688, year = {2025}, author = {Harris, H and Kittur, J}, title = {Unlocking the potential of CRISPR-Cas9 for cystic fibrosis: A systematic literature review.}, journal = {Gene}, volume = {942}, number = {}, pages = {149257}, doi = {10.1016/j.gene.2025.149257}, pmid = {39832688}, issn = {1879-0038}, mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; }, abstract = {CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders.}, }
@article {pmid39826273, year = {2025}, author = {Pal, T and Liu, Z and Chen, J}, title = {CIMNE-CRISPR: A novel amplification-free diagnostic for rapid early detection of African Swine Fever Virus.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117154}, doi = {10.1016/j.bios.2025.117154}, pmid = {39826273}, issn = {1873-4235}, support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; }, mesh = {*African Swine Fever Virus/isolation & purification/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology/blood ; *Biosensing Techniques/methods ; *Limit of Detection ; CRISPR-Cas Systems ; Magnetite Nanoparticles/chemistry ; DNA, Viral/analysis/genetics ; Reproducibility of Results ; }, abstract = {African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe3O4@SiO2 core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10[4] copies/μL and a limit of quantification (LOQ) of 4.2 × 10[5] copies/μL, with a dynamic range spanning 10[5] to 10[10] copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10[5] copies/μL of ASFV DNA even with high mutant concentrations (10[13] copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.}, }
@article {pmid39823857, year = {2025}, author = {Fan, X and Gao, Z and Ling, D and Wang, D and Cui, Y and Du, H and Zhou, X}, title = {The dCas9/crRNA linked immunological assay (dCLISA) for sensitive, accurate, and facile drug resistance gene analysis.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117147}, doi = {10.1016/j.bios.2025.117147}, pmid = {39823857}, issn = {1873-4235}, mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; Humans ; *Biosensing Techniques/methods ; *Staphylococcal Infections/microbiology/immunology ; *CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics ; Immunoassay/methods ; Drug Resistance, Bacterial/genetics ; }, abstract = {The rapid and reliable diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) is essential for preventing the spread of MRSA infections and guiding therapeutic strategies. However, there is still a huge challenge in further simplifying MRSA detection procedures and improving detection selectivity to reduce false-positive results. In this study, we developed a derivative CRISPR-associated protein 9/CRISPR-derived RNA Linked Immunological Assay (dCLISA) for the sensitive and specific detection of MRSA. This technique utilizes two dCas9/crRNA complexes as specific targeting agents and employs a color reaction mediated by a hybridization chain reaction for signal output. The dCLISA method offers certain benefits compared to monoclonal antibodies in traditional immunoassays, primarily due to its capacity to selectively interact with target gene and its high sensitivity from the hybridization chain reaction process. Therefore, the minimum detectable concentration of dCLISA was 8.5 cfu/mL. Unlike traditional gene analysis approaches, target gene sequences in cell lysates can be directly detected by dCLISA within 60 min with high sensitivity without genomic material extraction. In addition, the absorbance intensity of the MRSA cell lysate was significantly higher than that of methicillin-susceptible S. aureus (MSSA) indicates the clinical application potential. This study demonstrates that the dCLISA is a simple, rapid, sensitive, and specific method, which can be directly used at the point of care to analyze drug resistance in bacteria, including MRSA. Moreover, dCLISA can be utilized for other bacteria detection by merely modifying the crRNA sequence.}, }
@article {pmid39799696, year = {2025}, author = {Jiang, Y and Li, Y and Zheng, D and Du, X and Yang, H and Wang, C and Zhao, M and Xiao, H and Zhang, L and Li, X and Shi, S}, title = {Nano-polymeric platinum activates PAR2 gene editing to suppress tumor metastasis.}, journal = {Biomaterials}, volume = {317}, number = {}, pages = {123090}, doi = {10.1016/j.biomaterials.2025.123090}, pmid = {39799696}, issn = {1878-5905}, mesh = {*Receptor, PAR-2/metabolism/genetics ; Humans ; Animals ; *Platinum/chemistry/pharmacology/therapeutic use ; *Neoplasm Metastasis ; *Gene Editing ; Cell Line, Tumor ; Mice ; Cell Movement/drug effects ; CRISPR-Cas Systems ; Polymers/chemistry ; Mice, Nude ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/therapeutic use ; Female ; Nanoparticles/chemistry ; }, abstract = {Metastasis as the hallmark of cancer preferentially contributes to tumor recurrence and therapy resistance, aggrandizing the lethality of patients with cancer. Despite their robust suppressions of tumor progression, chemotherapeutics failed to attenuate cancer cell migration and even triggered pro-metastatic effects. In parallel, protease-activated receptor 2 (PAR2), a member of the G protein-coupled receptor subfamily, actively participates in cancer metastasis via multiple signal transduction pathways. CRISPR/Cas9 that is a dominating genome editing tool can evoke PAR2 knockout to inhibit cancer metastasis. However, the absence of valid delivery systems largely limits its efficacy. Herein, we nanosized polymeric platinum (NanoPt) as therapeutical drug carries to deliver CRISPR/Cas9 to elicit genome editing of PAR2, which drastically augmented anti-metastatic effects and alleviated systematic toxicity of platinum-based treatment in vitro and in vivo. More importantly, the NanoPt@Cas9-PAR2 initiated PAR2 deficiency to mechanistically attenuate EMT process and ferroptosis via RAGE/ERK signalling, consequently preventing cancer cell migration. Our findings indicate that NanoPt@Cas9-PAR2 that mitigated PAR2 signalling and cytotoxic effects of platinum could be a safe and powerful all-in-one combinatorial strategy for cancer treatment.}, }
@article {pmid38984870, year = {2025}, author = {Lu, J and Fu, B and Zhu, Z and Yan, C and Guan, F and Wang, P and Yu, P}, title = {Enhancing the production of L-proline in recombinant Escherichia coli BL21 by metabolic engineering.}, journal = {Preparative biochemistry & biotechnology}, volume = {55}, number = {2}, pages = {187-195}, doi = {10.1080/10826068.2024.2378104}, pmid = {38984870}, issn = {1532-2297}, mesh = {*Proline/metabolism ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Glucose/metabolism ; *CRISPR-Cas Systems ; *Fermentation ; Gene Editing/methods ; Escherichia coli Proteins/genetics/metabolism ; Batch Cell Culture Techniques ; }, abstract = {L-proline is widely used in the fields of food, medicine and agriculture, and is also an important raw material for the synthesis of trans-4-hydroxy-L-proline. In this study, enhancing the production of L-proline by metabolic engineering was investigated. Three genes, proB, proA and proC, were introduced into Escherichia coli BL21 by molecular biology technology to increase the metabolic flow of L-proline from glucose. The genes putP and proP related to the proline transfer were knocked out by CRISPR/Cas9 gene editing technology to weaken the feedback inhibition of proB to increase the production of L-proline. The fermentation curves of the engineered strain at different glucose concentrations were determined, and a glucose concentration of 10 g/L was chosen to expand the batch culture to 1 L shake flask. Ultimately, through these efforts, the titer of L-proline reached 832.19 mg/L in intermittent glucose addition fermentation in a 1 L shake flask.}, }
@article {pmid39889868, year = {2025}, author = {Zhao, Q and Wei, L and Chen, Y}, title = {From bench to bedside: Developing CRISPR/Cas-based therapy for ocular diseases.}, journal = {Pharmacological research}, volume = {}, number = {}, pages = {107638}, doi = {10.1016/j.phrs.2025.107638}, pmid = {39889868}, issn = {1096-1186}, abstract = {Vision-threatening disorders, including both hereditary and multifactorial ocular diseases, necessitate innovative therapeutic approaches. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has emerged as a promising tool for treating ocular diseases through gene editing and expression regulation. This system has contributed to the development of representative disease models, including animal models, organoids, and cell lines, thereby facilitating investigations into the pathogenesis of disease-related genes. Besides, therapeutic applications of CRISPR/Cas have been extensively explored in preclinical in vitro and in vivo studies, targeting various ocular conditions, such as retinitis pigmentosa, Leber congenital amaurosis, Usher syndrome, fundus neovascular diseases, glaucoma, and corneal diseases. Recent advancements have demonstrated the technology's potential to restore cellular homeostasis and alleviate disease phenotypes, thereby prompting a variety of clinical trials. To date, active trials include treatments for primary open angle glaucoma with MYOC mutations, refractory herpetic viral keratitis, CEP290-associated inherited retinal degenerations, neovascular age-related macular degeneration, and retinitis pigmentosa with RHO mutations. However, challenges remain, primarily concerning off-target effects, immunogenicity, ethical considerations, and regulatory particularity. To reach higher safety and efficiency before truly transitioning from bench to bedside, future research should concentrate on improving the specificity and efficacy of Cas proteins, optimizing delivery vectors, and broadening the applicability of therapeutic targets. This review summarizes the application strategies and delivery methods of CRISPR/Cas, discusses recent progress in CRISPR/Cas-based disease models and therapies, and provides an overview of the landscape of clinical trials. Current obstacles and future directions regarding the bench-to-bedside transition are also discussed.}, }
@article {pmid39887239, year = {2025}, author = {Chen, P-R and Wei, Y and Li, X and Yu, H-Y and Wang, S-G and Yuan, X-Z and Xia, P-F}, title = {Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {e0003125}, doi = {10.1128/aem.00031-25}, pmid = {39887239}, issn = {1098-5336}, abstract = {CRISPR-Cas systems are transforming precision medicine with engineered probiotics as next-generation diagnostics and therapeutics. To promote human health and treat disease, engineering probiotic bacteria demands maximal versatility to enable non-natural functionalities while minimizing undesired genomic interferences. Here, we present a streamlined prime editing approach tailored for probiotic Escherichia coli Nissle 1917 utilizing only essential genetic modules, including Cas9 nickase from Streptococcus pyogenes, a codon-optimized reverse transcriptase, and a prime editing guide RNA, and an optimized workflow with longer induction. As a result, we achieved all types of prime editing in every individual round of experiments with efficiencies of 25.0%, 52.0%, and 66.7% for DNA deletion, insertion, and substitution, respectively. A comprehensive evaluation of off-target effects revealed a significant reduction in unintended mutations, particularly in comparison to two different base editing methods. Leveraging the prime editing system, we inserted a unique DNA sequence to barcode the edited strain and established an antibiotic-resistance-gene-free platform to enable non-natural functionalities. Our prime editing strategy presents a CRISPR-Cas system that can be readily implemented in any laboratories with the basic CRISPR setups, paving the way for future innovations in engineered probiotics.IMPORTANCEOne ultimate goal of gene editing is to introduce designed DNA variations at specific loci in living organisms with minimal unintended interferences in the genome. Achieving this goal is especially critical for creating engineered probiotics as living diagnostics and therapeutics to promote human health and treat diseases. In this endeavor, we report a customized prime editing system for precision engineering of probiotic Escherichia coli Nissle 1917. With such a system, we developed a barcoding system for tracking engineered strains, and we built an antibiotic-resistance-gene-free platform to enable non-natural functionalities. We provide not only a powerful gene editing approach for probiotic bacteria but also new insights into the advancement of innovative CRISPR-Cas systems.}, }
@article {pmid39887096, year = {2025}, author = {Brusson, M and Miccio, A}, title = {[A CRISPR/Cas approach to β-haemoglobinopathies].}, journal = {Medecine sciences : M/S}, volume = {41}, number = {1}, pages = {33-39}, doi = {10.1051/medsci/2024191}, pmid = {39887096}, issn = {1958-5381}, support = {ANR-10-IAHU-01//Agence nationale de la recherche/ ; 865797 DITSB//Conseil européen de la recherche/ ; subvention HORIZON-RIA EDITSCD n° 101057659//Commission européenne/ ; subvention 22206//AFM-Téléthon/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; *beta-Thalassemia/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; Hematopoietic Stem Cells/metabolism/physiology ; Hemoglobinopathies/therapy/genetics ; Animals ; }, abstract = {Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.}, }
@article {pmid39806065, year = {2025}, author = {Chen, X and Ghanizada, M and Mallajosyula, V and Sola, E and Capasso, R and Kathuria, KR and Davis, MM}, title = {Differential roles of human CD4[+] and CD8[+] regulatory T cells in controlling self-reactive immune responses.}, journal = {Nature immunology}, volume = {26}, number = {2}, pages = {230-239}, pmid = {39806065}, issn = {1529-2916}, support = {U19 AI057229/AI/NIAID NIH HHS/United States ; AI057229//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; }, mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CD8-Positive T-Lymphocytes/immunology ; *Forkhead Transcription Factors/metabolism ; *Granzymes/metabolism ; *Autoimmunity ; Palatine Tonsil/immunology ; Animals ; Autoantigens/immunology ; Mice ; CRISPR-Cas Systems ; }, abstract = {Here we analyzed the relative contributions of CD4[+] regulatory T cells expressing Forkhead box protein P3 (FOXP3) and CD8[+] regulatory T cells expressing killer cell immunoglobulin-like receptors to the control of autoreactive T and B lymphocytes in human tonsil-derived immune organoids. FOXP3 and GZMB respectively encode proteins FOXP3 and granzyme B, which are critical to the suppressive functions of CD4[+] and CD8[+] regulatory T cells. Using CRISPR-Cas9 gene editing, we were able to achieve a reduction of ~90-95% in the expression of these genes. FOXP3 knockout in tonsil T cells led to production of antibodies against a variety of autoantigens and increased the affinity of influenza-specific antibodies. By contrast, GZMB knockout resulted in an increase in follicular helper T cells, consistent with the ablation of CD8[+] regulatory T cells observed in mouse models, and a marked expansion of autoreactive CD8[+] and CD4[+] T cells. These findings highlight the distinct yet complementary roles of CD8[+] and CD4[+] regulatory T cells in regulating cellular and humoral responses to prevent autoimmunity.}, }
@article {pmid39707712, year = {2025}, author = {Abdulsalam, L and Mordecai, J and Ahmad, I}, title = {Non-viral gene therapy for Leber's congenital amaurosis: progress and possibilities.}, journal = {Nanomedicine (London, England)}, volume = {20}, number = {3}, pages = {291-304}, doi = {10.1080/17435889.2024.2443387}, pmid = {39707712}, issn = {1748-6963}, mesh = {Humans ; *Genetic Therapy/methods ; *Leber Congenital Amaurosis/therapy/genetics ; Animals ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; Genetic Vectors/genetics ; Mutation ; CRISPR-Cas Systems/genetics ; }, abstract = {Leber's congenital amaurosis (LCA) represents a set of rare and pervasive hereditary conditions of the retina that cause severe vision loss starting in early childhood. Targeted treatment intervention has become possible thanks to recent advances in understanding LCA genetic basis. While viral vectors have shown efficacy in gene delivery, they present challenges related to safety, low cargo capacity, and the potential for random genomic integration. Non-viral gene therapy is a safer and more flexible alternative to treating the underlying genetic mutation causing LCA. Non-viral gene delivery methods, such as inorganic nanoparticles, polymer-based delivery systems, and lipid-based nanoparticles, bypass the risks of immunogenicity and genomic integration, potentially offering a more versatile and personalized treatment for patients. This review explores the genetic background of LCA, emphasizing the mutations involved, and explores diverse non-viral gene delivery methods being developed. It also highlights recent studies on non-viral gene therapy for LCA in animal models and clinical trials. It presents future perspectives for gene therapy, including integrating emerging technologies like CRISPR-Cas9, interdisciplinary collaborations, personalized medicine, and ethical considerations.}, }
@article {pmid39333753, year = {2025}, author = {Tiwari, SK and Wong, WJ and Moreira, M and Pasqualini, C and Ginhoux, F}, title = {Induced pluripotent stem cell-derived macrophages as a platform for modelling human disease.}, journal = {Nature reviews. Immunology}, volume = {25}, number = {2}, pages = {108-124}, pmid = {39333753}, issn = {1474-1741}, mesh = {Humans ; *Induced Pluripotent Stem Cells/immunology/cytology ; *Macrophages/immunology ; *Cell Differentiation/immunology ; Animals ; CRISPR-Cas Systems ; Organoids/immunology ; }, abstract = {Macrophages are innate immune cells that are present in essentially all tissues, where they have vital roles in tissue development, homeostasis and pathogenesis. The importance of macrophages in tissue function is reflected by their association with various human diseases, and studying macrophage functions in both homeostasis and pathological tissue settings is a promising avenue for new targeted therapies that will improve human health. The ability to generate macrophages from induced pluripotent stem (iPS) cells has revolutionized macrophage biology, with the generation of iPS cell-derived macrophages (iMacs) providing unlimited access to genotype-specific cells that can be used to model various human diseases involving macrophage dysregulation. Such disease modelling is achieved by generating iPS cells from patient-derived cells carrying disease-related mutations or by introducing mutations into iPS cells from healthy donors using CRISPR-Cas9 technology. These iMacs that carry disease-related mutations can be used to study the aetiology of the particular disease in vitro. To achieve more physiological relevance, iMacs can be co-cultured in 2D systems with iPS cell-derived cells or in 3D systems with iPS cell-derived organoids. Here, we discuss the studies that have attempted to model various human diseases using iMacs, highlighting how these have advanced our knowledge about the role of macrophages in health and disease.}, }
@article {pmid39039203, year = {2025}, author = {Naidoo, K and Oliver, SV}, title = {Gene drives: an alternative approach to malaria control?.}, journal = {Gene therapy}, volume = {32}, number = {1}, pages = {25-37}, pmid = {39039203}, issn = {1476-5462}, mesh = {*Malaria/prevention & control ; Animals ; *Gene Drive Technology/methods ; Humans ; *Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; Gene Editing/methods ; Culicidae/genetics ; }, abstract = {Genetic modification for the control of mosquitoes is frequently touted as a solution for a variety of vector-borne diseases. There has been some success using non-insecticidal methods like sterile or incompatible insect techniques to control arbovirus diseases. However, control by genetic modifications to reduce mosquito populations or create mosquitoes that are refractory to infection with pathogens are less developed. The advent of CRISPR-Cas9-mediated gene drives may advance this mechanism of control. In this review, use and progress of gene drives for vector control, particularly for malaria, is discussed. A brief history of population suppression and replacement gene drives in mosquitoes, rapid advancement of the field over the last decade and how genetic modification fits into the current scope of vector control are described. Mechanisms of alternative vector control by genetic modification to modulate mosquitoes' immune responses and anti-parasite effector molecules as part of a combinational strategy to combat malaria are considered. Finally, the limitations and ethics of using gene drives for mosquito control are discussed.}, }
@article {pmid39885543, year = {2025}, author = {Jiang, J and Cienfuegos-Galletd, AV and Long, T and Peirano, G and Chu, T and Pitout, JDD and Kreiswirth, BN and Chen, L}, title = {Intricate interplay of CRISPR-Cas systems, anti-CRISPR proteins, and antimicrobial resistance genes in a globally successful multi-drug resistant Klebsiella pneumoniae clone.}, journal = {Genome medicine}, volume = {17}, number = {1}, pages = {9}, pmid = {39885543}, issn = {1756-994X}, mesh = {*Klebsiella pneumoniae/genetics/drug effects ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; *Bacterial Proteins/genetics ; Phylogeny ; Plasmids/genetics ; beta-Lactamases/genetics ; Genome, Bacterial ; Klebsiella Infections/microbiology ; Humans ; Anti-Bacterial Agents/pharmacology ; }, abstract = {BACKGROUND: Klebsiella pneumoniae is one of the most prevalent pathogens responsible for multiple infections in healthcare settings and the community. K. pneumoniae CG147, primarily including ST147 (the founder ST), ST273, and ST392, is one of the most globally successful MDR clone linked to various carbapenemases.
METHODS: One hundred and one CG147 strains were sequenced and additional 911 publicly available CG147 genome sequences were included for analysis. The molecular epidemiology, population structure, and time phylogeny were investigated. The virulome, resistome, and mobilome were analyzed, and the recombination in the capsular region was studied. The CRISPR-Cas and anti-CRISPR were identified. The interplay between CRISPR-Cas, anti-CRISPR, and carbapenemase-encoding plasmids was analyzed and experimentally validated.
RESULTS: We analyzed 1012 global CG147 genomes, with 80.4% encoding at least one carbapenemase (NDM [529/1012, 52.3%], OXA-48-like [182/1012, 17.7%], and KPC [105/1012, 10.4%]). Surprisingly, almost all CG147 strains (99.7%, 1009/1,012) harbor a chromosomal type I-E CRISPR-Cas system, with 41.8% (423/1012) containing an additional plasmid-borne type IV-A3 CRISPR-Cas system, and both target IncF plasmids, e.g., the most prevalent KPC-encoding pKpQIL-like plasmids. We found the presence of IV-A3 CRISPR-Cas system showed a negative correlation with the presence of KPC. Interestingly, a prophage-encoding anti-CRISPR AcrIE8.1 and a plasmid-borne anti-CRISPR AcrIE9.2 were detected in 40.1% (406/1012) and 54.2% (548/1012) of strains, respectively, which displayed positive correlations with the presence of a carbapenemase. Plasmid transfer experiments confirmed that the I-E and IV-A3 CRISPR-Cas systems significantly decreased (p < 0.001) KPC-encoding pKpQIL plasmid conjugation frequencies, while the AcrIE8.1 and AcrIE9.2 significantly increased (p < 0.001) pKpQIL conjugation frequencies and protected plasmids from elimination by CRISPR-Cas I-E system.
CONCLUSIONS: Our results indicated a complex interplay between CRISPR-Cas, anti-CRISPR, and mobile genetic elements that shape the evolution of CG147. Our findings advance the understanding of multi-drug resistance mechanisms and will aid in preventing the emergence of future MDR clones.}, }
@article {pmid39885339, year = {2025}, author = {Low, SJ and O'Neill, M and Kerry, WJ and Wild, N and Krysiak, M and Nong, Y and Azzato, F and Hor, E and Williams, L and Taiaroa, G and Steinig, E and Pasricha, S and Williamson, DA}, title = {PathoGD: an integrative genomics approach to primer and guide RNA design for CRISPR-based diagnostics.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {147}, pmid = {39885339}, issn = {2399-3642}, support = {GA-F3791196-5514//State Government of Victoria (Victorian Government)/ ; PO4932//Department of Health, Australian Government (Department of Health)/ ; }, mesh = {*Genomics/methods ; *Streptococcus pyogenes/genetics ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Neisseria gonorrhoeae/genetics/isolation & purification ; Humans ; DNA Primers/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Critical to the success of CRISPR-based diagnostic assays is the selection of a diagnostic target highly specific to the organism of interest, a process often requiring iterative cycles of manual selection, optimisation, and redesign. Here we present PathoGD, a bioinformatic pipeline for rapid and high-throughput design of RPA primers and gRNAs for CRISPR-Cas12a-based pathogen detection. PathoGD is fully automated, leverages publicly available sequences and is scalable to large datasets, allowing rapid continuous monitoring and validation of primer/gRNA sets to ensure ongoing assay relevance. We designed primers and gRNAs for five clinically relevant bacterial pathogens, and experimentally validated a subset of the designs for detecting Streptococcus pyogenes and/or Neisseria gonorrhoeae in assays with and without pre-amplification. We demonstrated high specificity of primers and gRNAs designed, with minimal off-target signal observed for all combinations. We anticipate PathoGD will be an important resource for assay design for current and emerging pathogens. PathoGD is available on GitHub at https://github.com/sjlow23/pathogd .}, }
@article {pmid39885211, year = {2025}, author = {Cheng, ZH and Luo, XY and Yu, SS and Min, D and Zhang, SX and Li, XF and Chen, JJ and Liu, DF and Yu, HQ}, title = {Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1166}, pmid = {39885211}, issn = {2041-1723}, mesh = {*SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems ; Humans ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/metabolism/genetics ; Influenza A virus/genetics ; COVID-19/virology/diagnosis ; Bacterial Proteins/genetics/metabolism ; Saliva/virology ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; Heparin/metabolism ; Molecular Diagnostic Techniques/methods ; }, abstract = {The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing.}, }
@article {pmid39885149, year = {2025}, author = {Jin, W and Deng, Y and La Marca, JE and Lelliott, EJ and Diepstraten, ST and König, C and Tai, L and Snetkova, V and Dorighi, KM and Hoberecht, L and Hedditch, MG and Whelan, L and Healey, G and Fayle, D and Lau, K and Potts, MA and Chen, MZ and Johnston, APR and Liao, Y and Shi, W and Kueh, AJ and Haley, B and Fortin, JP and Herold, MJ}, title = {Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {974}, pmid = {39885149}, issn = {2041-1723}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Lymphoma/genetics ; Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/genetics/metabolism ; Humans ; Gene Knockout Techniques/methods ; Red Fluorescent Protein ; Fibroblasts/metabolism ; Mice, Inbred C57BL ; }, abstract = {Cas12a is a next-generation gene editing tool that enables multiplexed gene targeting. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp. Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We demonstrate efficient single and multiplexed gene editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We also present compact, genome-wide Cas12a knockout libraries, with four crRNAs per gene encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrate the utility of these libraries across methodologies: in vitro enrichment and drop-out screening in lymphoma cells and immortalised fibroblasts, respectively, and in vivo screens to identify lymphoma-driving events. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas9-SAM) using primary T cells and fibroblasts. Our enAsCas12a mouse and accompanying crRNA libraries enhance genome engineering capabilities and complement current CRISPR technologies.}, }
@article {pmid39754719, year = {2025}, author = {Chu, SN and Soupene, E and Sharma, D and Sinha, R and McCreary, T and Hernandez, B and Shen, H and Wienert, B and Bowman, C and Yin, H and Lesch, BJ and Jia, K and Romero, KA and Kostamo, Z and Zhang, Y and Tran, T and Cordero, M and Homma, S and Hampton, JP and Gardner, JM and Conklin, BR and MacKenzie, TC and Sheehan, VA and Porteus, MH and Cromer, MK}, title = {Dual α-globin-truncated erythropoietin receptor knockin restores hemoglobin production in α-thalassemia-derived erythroid cells.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115141}, doi = {10.1016/j.celrep.2024.115141}, pmid = {39754719}, issn = {2211-1247}, support = {T32 AI125222/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; *alpha-Globins/genetics/metabolism ; *Erythroid Cells/metabolism ; *alpha-Thalassemia/genetics/metabolism ; *Receptors, Erythropoietin/metabolism/genetics ; *Gene Editing/methods ; *Hemoglobins/metabolism ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; Erythropoiesis/genetics ; Hematopoietic Stem Cells/metabolism ; beta-Globins/genetics/metabolism ; }, abstract = {The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.}, }
@article {pmid39745853, year = {2025}, author = {Wei, R and Yu, Z and Ding, L and Lu, Z and Yao, K and Zhang, H and Huang, B and He, M and Ma, L}, title = {Improved split prime editors enable efficient in vivo genome editing.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115144}, doi = {10.1016/j.celrep.2024.115144}, pmid = {39745853}, issn = {2211-1247}, mesh = {*Gene Editing/methods ; Animals ; *Dependovirus/genetics ; Mice ; Humans ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; }, abstract = {Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.}, }
@article {pmid39721023, year = {2025}, author = {Chen, C and Liao, Y and Zhu, M and Wang, L and Yu, X and Li, M and Peng, G}, title = {Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115105}, doi = {10.1016/j.celrep.2024.115105}, pmid = {39721023}, issn = {2211-1247}, mesh = {Animals ; *Cell Lineage ; Mice ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Cell Differentiation ; Endodeoxyribonucleases/metabolism/genetics ; Humans ; Embryoid Bodies/cytology/metabolism ; Bacterial Proteins/metabolism/genetics ; }, abstract = {Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.}, }
@article {pmid39300230, year = {2025}, author = {Li, G and Cheng, Y and Yu, J and Zhu, Y and Ma, H and Zhou, Y and Pu, Z and Zhu, G and Yuan, Y and Zhang, Z and Zhou, X and Tian, K and Qiao, J and Hu, X and Chen, XX and Ji, Q and Huang, X and Ma, B and Yao, Y}, title = {Compact RNA editors with natural miniature Cas13j nucleases.}, journal = {Nature chemical biology}, volume = {21}, number = {2}, pages = {280-290}, pmid = {39300230}, issn = {1552-4469}, support = {2023YFC3402402//Ministry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)/ ; 02020200-K02013008//Zhejiang University (ZJU)/ ; }, mesh = {Animals ; Mice ; *Dependovirus/genetics ; Humans ; RNA Editing ; Proprotein Convertase 9/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism/genetics ; RNA/genetics/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeats-Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3[D65N] and SCN9A[R896Q], to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.}, }
@article {pmid39884612, year = {2025}, author = {He, D and Zhao, S and Wang, F and Wu, B and Wei, F and Zhao, Y and Wei, X and Ren, H and Zhang, M and Fan, Y and Zhang, J and Yu, S and Tang, Y and Diao, Y}, title = {H9N2 avian influenza virus diagnostics utilizing specific high-sensitivity enzymatic molecular system termed RPA-based CRISPR-Cas13a.}, journal = {International journal of biological macromolecules}, volume = {}, number = {}, pages = {140474}, doi = {10.1016/j.ijbiomac.2025.140474}, pmid = {39884612}, issn = {1879-0003}, abstract = {H9N2 avian influenza virus (AIV), a major pathogen causing respiratory infections in poultry, poses a significant threat to the poultry industry and human health. Early detection and control of H9N2 infections are essential for minimizing economic losses and preventing potential zoonotic transmission. A novel CRISPR-Cas family member called CRISPR-Cas13a comprises the CRISPR RNA (crRNA) and Cas13a nuclease. Through the crRNA-based reprogramming of Cas13a, a platform for sensing RNAs specifically is available. In this study, we developed a RPA-based CRISPR-Cas13a diagnostic method for rapid detection of the H9N2 AIV. The results demonstrated that at a limit of 10 copies/μL and 10[2] copies/μL could be detected within 50 min, by fluorescence detection and lateral flow strip, respectively, offering a highly sensitive method for H9N2 detection. This method exhibited excellent specificity, distinguishing H9N2 from other pathogens. Furthermore, the RPA-Cas13a-based detection system was tested on clinical samples, showing comparable performance to RT-qPCR. The detection results were visualized using either lateral flow assays or fluorescence, making it a suitable tool for on-site, field-deployable diagnostics. In a word, this RPA-Cas13a diagnostic approach offers high reliability, sensitivity, and specificity, with promising potential for rapidly detecting H9N2 and other viral pathogens in clinical and food safety applications.}, }
@article {pmid39884405, year = {2025}, author = {S, BR and Dhar, R and Devi, A}, title = {Exosome-mediated CRISPR/Cas delivery: A cutting-edge frontier in cancer gene therapy.}, journal = {Gene}, volume = {}, number = {}, pages = {149296}, doi = {10.1016/j.gene.2025.149296}, pmid = {39884405}, issn = {1879-0038}, abstract = {Cancer is considered the second most common disease globally. In the past few decades, many approaches have been proposed for cancer treatment. One among those is targeted therapy using CRISPR-Cas system which plays an irreplaceable role in translational research through gene editing. However, due to its inability to cope with specific targeting, off-target effects, and limited tumor penetration, it is very challenging to use this approach in cancer studies. To increase its efficacy, CRISPR components are engineered into the extracellular vesicles (EVs), especially exosomes (a subpopulation of EVs). Exosomes have a significant role in cellular communication. Exosome-based CRISPR-Cas system transport for gene editing enhanced specificity, reduced off-target effects, and improved therapeutic potential. In this review, we highlighted the role of exosomes and the CRISPR-Cas system in cancer research, exosome-based CRISPR delivery for cancer treatment, and its future orientation.}, }
@article {pmid39883775, year = {2025}, author = {Koeppel, J and Ferreira, R and Vanderstichele, T and Riedmayr, LM and Peets, EM and Girling, G and Weller, J and Murat, P and Liberante, FG and Ellis, T and Church, GM and Parts, L}, title = {Randomizing the human genome by engineering recombination between repeat elements.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6733}, pages = {eado3979}, doi = {10.1126/science.ado3979}, pmid = {39883775}, issn = {1095-9203}, mesh = {Humans ; *Genome, Human ; *Repetitive Sequences, Nucleic Acid ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombination, Genetic ; Translocation, Genetic ; Sequence Deletion ; Gene Rearrangement ; Chromosome Inversion ; Genes, Essential ; Genetic Engineering/methods ; }, abstract = {We lack tools to edit DNA sequences at scales necessary to study 99% of the human genome that is noncoding. To address this gap, we applied CRISPR prime editing to insert recombination handles into repetitive sequences, up to 1697 per cell line, which enables generating large-scale deletions, inversions, translocations, and circular DNA. Recombinase induction produced more than 100 stochastic megabase-sized rearrangements in each cell. We tracked these rearrangements over time to measure selection pressures, finding a preference for shorter variants that avoided essential genes. We characterized 29 clones with multiple rearrangements, finding an impact of deletions on expression of genes in the variant but not on nearby genes. This genome-scrambling strategy enables large deletions, sequence relocations, and the insertion of regulatory elements to explore genome dispensability and organization.}, }
@article {pmid39883010, year = {2025}, author = {Zhang, W and Zhong, Y and Wang, J and Zou, G and Chen, Q and Liu, C}, title = {Direct repeat region 3' end modifications regulate Cas12a activity and expand its applications.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkaf040}, pmid = {39883010}, issn = {1362-4962}, support = {22307150//National Natural Science Foundation of China/ ; A2303012//Shenzhen Medical Research Fund/ ; 2024A1515012319//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20230807110315032//Shenzhen Science and Technology Program/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; Alkaline Phosphatase/metabolism/genetics ; alpha-Fetoproteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins/metabolism/genetics ; Phosphorylation ; Immunoglobulin G ; DNA/metabolism/genetics/chemistry ; Herpesvirus 4, Human/genetics ; }, abstract = {CRISPR-Cas12a technology has transformative potential, but as its applications grow, enhancing its inherent functionalities is essential to meet diverse demands. Here, we reveal a regulatory mechanism for LbCas12a through direct repeat (DR) region 3' end modifications and de-modifications, which can regulate LbCas12a's cis- and trans-cleavage activities. We extensively explored the effects of introducing phosphorylation, DNA, photo-cleavable linker, DNA modifications at the DR 3' end on LbCas12a's functionality. We find that the temporary inhibitory function of Cas12a can be reactivated by DR 3' end modification corresponding substances, such as alkaline phosphatase (ALP), immunoglobulin G (IgG), alpha-fetoprotein (AFP), DNA exonucleases, ultraviolet radiation, and DNA glycosylases, which greatly expand the scope of application of Cas12a. Clinical applications demonstrated promising results in ALP, AFP, and trace Epstein-Barr virus detection compared to gold standard methods. Our research provides valuable insights into regulating LbCas12a activity through direct modification of DR and significantly expands its potential clinical detection targets, paving the way for future universal clustered regularly interspaced short palindromic repeats (CRISPR) diagnostic strategies.}, }
@article {pmid39880939, year = {2025}, author = {Wang, W and Pan, Q and Tian, B and Yu, Z and Davidson, D and Bai, G and Akhunova, A and Trick, H and Akhunov, E}, title = {Non-additive dosage-dependent effects of TaGS3 gene editing on grain size and weight in wheat.}, journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik}, volume = {138}, number = {2}, pages = {38}, pmid = {39880939}, issn = {1432-2242}, support = {2021-67013-34174//National Institute of Food and Agriculture/ ; 2020-67013-30906//National Institute of Food and Agriculture/ ; 2022-68013-36439//National Institute of Food and Agriculture/ ; INV-004430/GATES/Bill & Melinda Gates Foundation/United States ; }, mesh = {*Triticum/genetics/growth & development ; *Gene Editing/methods ; *Edible Grain/genetics/growth & development ; *CRISPR-Cas Systems ; Alleles ; Phenotype ; Plant Proteins/genetics ; Seeds/growth & development/genetics ; Gene Dosage ; Genes, Plant ; Plants, Genetically Modified/growth & development/genetics ; Loss of Function Mutation ; }, abstract = {Loss-of-function mutations induced by CRISPR-Cas9 in the TaGS3 gene homoeologs show non-additive dosage-dependent effects on grain size and weight and have potential utility for increasing grain yield in wheat. The grain size in cereals is one of the component traits contributing to yield. Previous studies showed that loss-of-function (LOF) mutations in GS3, encoding Gγ subunit of the multimeric G protein complex, increase grain size and weight in rice. While an association between allelic variation in the GS3 homologs of wheat and grain weight/size has been detected previously, the effects of LOF alleles at TaGS3 on these traits remain unknown. We used genome editing to create TaGS3 mutant lines with varying LOF homeo-allele dosages. Contrary to the results obtained in rice, editing all three TaGS3 homoeologous copies resulted in a significant decrease in grain length (4.4%), width (3.4%), grain area (7.3%) and weight (7.5%), without affecting the number of grains per spike. Compared to the wild type, the highest increase in grain weight (up to 9.6%) and area (up to 5.0%) was observed in homozygous mutants with one or two genomes carrying LOF homeo-alleles, suggesting non-additive suppressive effects of TaGS3 on grain size and weight in wheat. Our results suggest that the regulatory effects of GS3 homologs in wheat and rice have diverged. The newly developed LOF homeo-alleles of TaGS3 expand the set of CRISPR-Cas9-induced variants of yield component genes that have potential to increase grain weight in wheat.}, }
@article {pmid39880685, year = {2025}, author = {van Dongen, JE and Segerink, LI}, title = {Building the Future of Clinical Diagnostics: An Analysis of Potential Benefits and Current Barriers in CRISPR/Cas Diagnostics.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00816}, pmid = {39880685}, issn = {2161-5063}, abstract = {Advancements in molecular diagnostics, such as polymerase chain reaction and next-generation sequencing, have revolutionized disease management and prognosis. Despite these advancements in molecular diagnostics, the field faces challenges due to high operational costs and the need for sophisticated equipment and highly trained personnel besides having several technical limitations. The emergent field of CRISPR/Cas sensing technology is showing promise as a new paradigm in clinical diagnostics, although widespread clinical adoption remains limited. This perspective paper discusses specific cases where CRISPR/Cas technology can surmount the challenges of existing diagnostic methods by stressing the significant role that CRISPR/Cas technology can play in revolutionizing clinical diagnostics. It underscores the urgency and importance of addressing the technological and regulatory hurdles that must be overcome to harness this technology effectively in clinical laboratories.}, }
@article {pmid39603170, year = {2025}, author = {Ali, N and Singh, S and Garg, R}, title = {Unlocking crops' genetic potential: Advances in genome and epigenome editing of regulatory regions.}, journal = {Current opinion in plant biology}, volume = {83}, number = {}, pages = {102669}, doi = {10.1016/j.pbi.2024.102669}, pmid = {39603170}, issn = {1879-0356}, mesh = {*Crops, Agricultural/genetics ; *Gene Editing/methods ; *Genome, Plant/genetics ; Epigenesis, Genetic ; Epigenome/genetics ; CRISPR-Cas Systems ; Plant Breeding/methods ; }, abstract = {Genome editing tools could precisely and efficiently target plant genomes leading to the development of improved crops. Besides editing the coding regions, researchers can employ editing technologies to target specific gene regulatory elements or modify epigenetic marks associated with distal regulatory regions, thereby regulating gene expression in crops. This review outlines several prominent genome editing technologies, including CRISPR-Cas9, TALENs, and ZFNs and recent advancements. The applications for genome and epigenome editing especially of regulatory regions in crop plants is also discussed, including efforts to enhance abiotic stress tolerance, yield, disease resistance and plant phenotype. Additionally, the review addresses the potential of epigenetic modifications, such as DNA methylation and histone modifications, to alter gene expression for crop improvement. Finally, the limitations and future scope of utilizing various genome editing tools to manipulate regulatory elements for gene regulation to unlock the full potential of these tools in plant breeding has been discussed.}, }
@article {pmid39880501, year = {2025}, author = {Tian, Y and Chen, J and Chen, F and Xu, J and Huang, L and Peng, L and Li, H and Shi, K}, title = {Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.}, journal = {Analytica chimica acta}, volume = {1341}, number = {}, pages = {343662}, doi = {10.1016/j.aca.2025.343662}, pmid = {39880501}, issn = {1873-4324}, mesh = {*Malathion/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques ; *Aptamers, Nucleotide/chemistry ; *RNA, Guide, CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {BACKGROUND: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.
RESULTS: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.
SIGNIFICANCE: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.}, }
@article {pmid39880493, year = {2025}, author = {Wang, S and Shen, X and Chen, G and Zhang, W and Tan, B}, title = {Application and development of CRISPR-Cas12a methods for the molecular diagnosis of cancer: A review.}, journal = {Analytica chimica acta}, volume = {1341}, number = {}, pages = {343603}, doi = {10.1016/j.aca.2024.343603}, pmid = {39880493}, issn = {1873-4324}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; Molecular Diagnostic Techniques/methods ; }, abstract = {Rapid, sensitive, and specific molecular detection methods are crucial for diagnosing, treating and prognosing cancer patients. With advancements in biotechnology, molecular diagnostic technology has garnered significant attention as a fast and accurate method for cancer diagnosis. CRISPR-Cas12a (Cpf1), an important CRISPR-Cas family member, has revolutionized the field of molecular diagnosis since its introduction. CRISPR-Cas technologies are a new generation of molecular tools that are widely used in the detection of pathogens, cancers, and other diseases. Liquid biopsy methods based on CRISPR-Cas12a have demonstrated remarkable success in cancer diagnosis, encompassing the detection of DNA mutations, DNA methylation, tumor-related viruses, and non-nucleic acid molecule identification. This review systematically discusses the developmental history, key technologies, and principles of CRISPR-Cas12a-based molecular diagnostic techniques and their applications in cancer diagnosis. This review has also discussed the future development directions of CRISPR-Cas12a, aiming for it to become a reliable new technology that can be used in clinical application.}, }
@article {pmid39880137, year = {2025}, author = {Yang, S and Wei, Y and Quansah, E and Zhang, Z and Da, W and Wang, B and Wang, K and Sun, D and Tao, Z and Zhang, C}, title = {Cas12a Is Competitive for Gene Editing in the Malaria parasites.}, journal = {Microbial pathogenesis}, volume = {}, number = {}, pages = {107340}, doi = {10.1016/j.micpath.2025.107340}, pmid = {39880137}, issn = {1096-1208}, abstract = {Malaria, caused by the Plasmodium parasites, has always been one of the worst infectious diseases that threaten human health, making it necessary for us to study the genetic function and physiological mechanisms of Plasmodium parasites from the molecular level to find more effective ways of addressing the increasingly pressing threat. The CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) is an RNA-guided adaptive immune system, which has been extensively developed and used as a genome editing tool in many organisms, including Plasmodium parasites. However, due to the physiological characteristics and special genomic characteristics of Plasmodium parasites, most of the tools currently used for genome editing of Plasmodium parasites have not met expectations. CRISPR-Cas12a (also known as Cpf1), one of the CRISPR-Cas systems, has attracted considerable attention because of its characteristics of being used for biological diagnosis and multiple genome editing. Recent studies have shown that its unique properties fit the genetic makeup of Plasmodium parasites making it a promising tool for gene editing in these parasites. In this review, we have summarized the relevant content of the Cas12 family, especially the frequently used Cas12a, its advantages for gene editing, and the application prospects in Plasmodium parasites.}, }
@article {pmid39879084, year = {2025}, author = {Eom, H and Choi, YJ and Nandre, R and Kim, M and Oh, YL and Kim, S and Nakazawa, T and Honda, Y and Ro, HS}, title = {Targeted insertion of heterogenous DNA using Cas9-gRNA ribonucleoprotein-mediated gene editing in Ganoderma lucidum.}, journal = {Bioengineered}, volume = {16}, number = {1}, pages = {2458376}, doi = {10.1080/21655979.2025.2458376}, pmid = {39879084}, issn = {2165-5987}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Reishi/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA Breaks, Double-Stranded ; DNA, Fungal/genetics/metabolism ; }, abstract = {Gene editing is emerging as a powerful tool for introducing novel functionalities in mushrooms. While CRISPR/Cas9-induced double-strand breaks (DSBs) typically rely on non-homologous end joining (NHEJ) for gene disruption, precise insertion of heterologous DNA in mushrooms is less explored. Here, we evaluated the efficacy of inserting donor DNAs (8-1008 bp) with or without homologous arms at Cas9-gRNA RNP-induced DSBs. Co-transformation of donor DNAs with RNP targeting the pyrG gene in Ganoderma lucidum yielded 184 transformants without homologous arms and 781 with 300-bp homologous arms (HR_donor DNAs). Restriction analysis and sequencing identified 122 hR_donor DNA transformants with complete donor DNA sequences, achieving 15.6% HDR efficiency (122/781), contrasting with 8 instances via NHEJ from the 184 transformants. These findings highlight the viability of HDR for precise genomic editing in mushrooms, enabling targeted modifications to enhance functionalities.}, }
@article {pmid39878871, year = {2025}, author = {Schöllkopf, AI and Almeida, L and Krammer, K and Rivero, CG and Liebl, W and Ehrenreich, A}, title = {Deletion of atypical type II restriction genes in Clostridium cellulovorans using a Cas9-based gene editing system.}, journal = {Applied microbiology and biotechnology}, volume = {109}, number = {1}, pages = {31}, pmid = {39878871}, issn = {1432-0614}, support = {161B0930//Bundesministerium für Bildung und Forschung/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Clostridium cellulovorans/genetics/metabolism ; Promoter Regions, Genetic ; Gene Deletion ; CRISPR-Associated Protein 9/genetics/metabolism ; Riboswitch/genetics ; Conjugation, Genetic ; Bacterial Proteins/genetics/metabolism ; }, abstract = {The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain. KEY POINTS: • Quantification of heterologous promoters enables rational choice for genetic engineering. • CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans. • Conjugation protocol and type II REase deletion enhance genetic accessibility.}, }
@article {pmid39878690, year = {2025}, author = {I Edvard Smith, C and Zain, R and Blomberg, P}, title = {[Gene editing is changing the treatment of hereditary diseases].}, journal = {Lakartidningen}, volume = {122}, number = {}, pages = {}, pmid = {39878690}, issn = {1652-7518}, mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *CRISPR-Cas Systems ; }, abstract = {Gene editing is a novel technology within gene therapy, which changes sequences in chromosomal DNA with precision. Even if there are alternative strategies, the Nobel Prize-winning CRISPR/Cas technology has become the dominating principle. During recent years base editing and prime editing, permitting editing without DNA double-strand breaks, have been developed. The first clinical gene editing results were reported in 2021; since then many patients have been treated, and recently the first treatment was approved as a novel therapy in the UK and later in USA and in EU. This update describes various aspects including methodological developments and safety.}, }
@article {pmid39878523, year = {2025}, author = {Xiao, X and Yang, S and Jiang, G and He, S}, title = {Current views and trends of nanomaterials as vectors for gene delivery since the 21st century: a bibliometric analysis.}, journal = {Nanomedicine (London, England)}, volume = {}, number = {}, pages = {1-16}, doi = {10.1080/17435889.2025.2457781}, pmid = {39878523}, issn = {1748-6963}, abstract = {BACKGROUND: Gene therapy is garnering increasing support due to its potential for a "once-delivered, lifelong benefit." The limitations of traditional gene delivery methods have spurred the advancement of bionanomaterials. Despite this progress, a thorough analysis of the evolution, current state, key contributors, focal studies, and future directions of nanomaterials in gene delivery remains absent.
METHODS: This study scrutinizes articles from the Web of Science, spanning 1 January 2 000, to 31 December 2023, employing various online tools for analysis and visualization.
RESULTS: The 21st century has witnessed consistent growth in scholarly work in this domain globally, with notable contributions from China and the US. At the same time, the Chinese Academy of Sciences (CAS), Harvard University, and Massachusetts Institute of Technology (MIT) have emerged as the most productive institutions, with CAS's academician Weihong Tan becoming the field's leading author. While drug delivery and nanoparticles (NPs) have been central themes for two decades, the research focus has shifted from modifying NPs and ultrafine particles to exploring polymer-hybrid NPs, mRNA vaccines, immune responses, green synthesis, and CRISPR/Cas tools.
CONCLUSIONS: This shift marks the transition from nanomaterials to bionanomaterials. The insights provided by this research offer a comprehensive overview of the field and valuable guidance for future investigations.}, }
@article {pmid39878334, year = {2025}, author = {Yuan, Y and Li, Y and Li, G and Lei, L and Huang, X and Li, M and Yao, Y}, title = {Intelligent Design of Lipid Nanoparticles for Enhanced Gene Therapeutics.}, journal = {Molecular pharmaceutics}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.molpharmaceut.4c00925}, pmid = {39878334}, issn = {1543-8392}, abstract = {Lipid nanoparticles (LNPs) are an effective delivery system for gene therapeutics. By optimizing their formulation, the physiochemical properties of LNPs can be tailored to improve tissue penetration, cellular uptake, and precise targeting. The application of these targeted delivery strategies within the LNP framework ensures efficient delivery of therapeutic agents to specific organs or cell types, thereby maximizing therapeutic efficacy. In the realm of genome editing, LNPs have emerged as a potent vehicle for delivering CRISPR/Cas components, offering significant advantages such as high in vivo efficacy. The incorporation of machine learning into the optimization of LNP platforms for gene therapeutics represents a significant advancement, harnessing its predictive capabilities to substantially accelerate the research and development process. This review highlights the dynamic evolution of LNP technology, which is expected to drive transformative progress in the field of gene therapy.}, }
@article {pmid39878102, year = {2025}, author = {Khosravi, S and Hinrichs, R and Rönspies, M and Haghi, R and Puchta, H and Houben, A}, title = {Epigenetic state and gene expression remain stable after CRISPR/Cas-mediated chromosomal inversions.}, journal = {The New phytologist}, volume = {}, number = {}, pages = {}, doi = {10.1111/nph.20403}, pmid = {39878102}, issn = {1469-8137}, support = {EpiChrom 031B1220B//Bundesministerium für Bildung und Forschung/ ; }, abstract = {The epigenetic state of chromatin, gene activity and chromosomal positions are interrelated in plants. In Arabidopsis thaliana, chromosome arms are DNA-hypomethylated and enriched with the euchromatin-specific histone mark H3K4me3, while pericentromeric regions are DNA-hypermethylated and enriched with the heterochromatin-specific mark H3K9me2. We aimed to investigate how the chromosomal location affects epigenetic stability and gene expression by chromosome engineering. Two chromosomal inversions of different sizes were induced using CRISPR/Cas9 to move heterochromatic, pericentric sequences into euchromatic regions. The epigenetic status of these lines was investigated using whole-genome bisulfite sequencing and chromatin immunoprecipitation. Gene expression changes following the induction of the chromosomal inversions were studied via transcriptome analysis. Both inversions had a minimal impact on the global distribution of histone marks and DNA methylation patterns, although minor epigenetic changes were observed across the genome. Notably, the inverted chromosomal regions and their borders retained their original epigenetic profiles. Gene expression analysis showed that only 0.5-1% of genes were differentially expressed genome-wide following the induction of the inversions. CRISPR/Cas-induced chromosomal inversions minimally affect epigenetic landscape and gene expression, preserving their profiles in subsequent generations.}, }
@article {pmid39877015, year = {2024}, author = {Fatkulin, AA and Chuksina, TA and Sorokina, NP and Smykov, IT and Kuraeva, EV and Masezhnaya, ES and Smagina, KA and Shkurnikov, MY}, title = {Comparative Analysis of Spacer Targets in CRISPR-Cas Systems of Starter Cultures.}, journal = {Acta naturae}, volume = {16}, number = {4}, pages = {81-85}, pmid = {39877015}, issn = {2075-8251}, abstract = {Dairy production facilities represent a unique ecological niche for bacteriophages of lactic acid bacteria. Throughout evolution, bacteria have developed a wide range of defense mechanisms against viral infections caused by bacteriophages. The CRISPR-Cas system is of particular interest due to its adaptive nature. It allows bacteria to acquire and maintain specific resistance to certain bacteriophages. In this study, we investigated the CRISPR-Cas systems of lactic acid bacteria. Special attention was paid to the specificity of the spacers in CRISPR cassettes. CRISPR-Cas systems were found in the genomes of 43% of the lactic acid bacteria studied. Additionally, only 13.1% of the total number of CRISPR cassette spacers matched bacteriophage genomes, indicating that many predicted spacers either lack known phage targets or are directed against other types of mobile genetic elements, such as plasmids.}, }
@article {pmid39876973, year = {2024}, author = {de Mello, DC and Menezes, JM and de Oliveira, ATF and Cristovão, MM and Kimura, ET and Fuziwara, CS}, title = {Modulating gene expression as a strategy to investigate thyroid cancer biology.}, journal = {Archives of endocrinology and metabolism}, volume = {68}, number = {Spec Issue}, pages = {e240073}, pmid = {39876973}, issn = {2359-4292}, mesh = {Humans ; *Thyroid Neoplasms/genetics ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Regulation, Neoplastic/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; }, abstract = {Modulating the expression of a coding or noncoding gene is a key tool in scientific research. This strategy has evolved methodologically due to advances in cloning approaches, modeling/algorithms in short hairpin RNA (shRNA) design for knockdown efficiency, and biochemical modifications in RNA synthesis, among other developments. Overall, these modifications have improved the ways to either reduce or induce the expression of a given gene with efficiency and facility for implementation in the lab. Allied with that, the existence of various human cell line models for cancer covering different histotypes and biological behaviors, especially for thyroid cancer, has helped improve the understanding of cancer biology. In this review, we cover the most frequently used current techniques for gene modulation in the thyroid cancer field, such as RNA interference (RNAi), short hairpin RNA (shRNA), and gene editing with CRISPR/Cas9 for inhibiting a target gene, and strategies to overexpress a gene, such as plasmid cloning and CRISPRa. Exploring the possibilities for gene modulation allows the improvement of the scientific quality of the studies and the integration of clinicians and basic scientists, leading to better development of translational research.}, }
@article {pmid39875802, year = {2025}, author = {Raudstein, M and Peñaranda, MMD and Kjærner-Semb, E and Grove, S and Morton, HC and Edvardsen, RB}, title = {Generation of IgM[+] B cell-deficient Atlantic salmon (Salmo salar) by CRISPR/Cas9-mediated IgM knockout.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {3599}, pmid = {39875802}, issn = {2045-2322}, mesh = {Animals ; *CRISPR-Cas Systems ; *Salmo salar/genetics/immunology ; *Immunoglobulin M/genetics/immunology ; *B-Lymphocytes/immunology/metabolism ; Gene Knockout Techniques ; }, abstract = {Infectious diseases pose significant challenges to Norwegian Atlantic salmon aquaculture. Vaccines are critical for disease prevention; however, a deeper understanding of the immune system is essential to improve vaccine efficacy. Immunoglobulin M (IgM) is the main antibody involved in the systemic immune response of teleosts, including Atlantic salmon. In this study, we used CRISPR/Cas9 technology to knock out the two IgM genes in Atlantic salmon. High-throughput sequencing revealed an average mutagenesis efficiency of 97% across both loci, with a predominance of frameshift mutations (78%). Gene expression analyses demonstrated significantly reduced membrane-bound IgM mRNA levels in head kidney and spleen tissues. Flow cytometry revealed a 78% reduction in IgM[+] B cells in peripheral blood, and Western blot analyses showed decreased IgM protein levels in serum. Notably, an upregulation of IgT mRNA was observed, suggesting a potential compensatory mechanism. This work presents the first application of CRISPR/Cas9 to disrupt an immune-related gene in the F0 generation of Atlantic salmon, and lays the foundation for generating a model completely lacking IgM[+] B cells which can be used to study the role of B cells and antibodies. This study has implications for advancing immune research in teleosts and for developing strategies to improve salmon health and welfare in aquaculture.}, }
@article {pmid39873837, year = {2025}, author = {Asa, H and Kuwabara, C and Matsumoto, K and Shigeta, R and Yamamoto, T and Masuda, Y and Yamada, T}, title = {Simultaneous site-directed mutagenesis for soybean ß-amyrin synthase genes via DNA-free CRISPR/Cas9 system using a single gRNA.}, journal = {Plant cell reports}, volume = {44}, number = {2}, pages = {40}, pmid = {39873837}, issn = {1432-203X}, support = {JPMJSP2119//JST SPRING/ ; }, mesh = {*Glycine max/genetics/enzymology ; *Mutagenesis, Site-Directed/methods ; *CRISPR-Cas Systems ; Intramolecular Transferases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Saponins/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Mutation/genetics ; Oleanolic Acid/analogs & derivatives/metabolism ; Gene Editing/methods ; }, abstract = {We generated soybean mutants related to two ß-amyrin synthase genes using DNA-free site-directed mutagenesis system. Our results suggested that one of the genes is predominant in the soyasaponin biosynthesis. Soyasaponins, which are triterpenoid saponins contained in soybean [Glycine max (L.) Merril], are responsible for the astringent aftertaste of soyfood, and their complete elimination from soybean seeds is a key challenge in the development of cultivars with improved taste. While the loss of function in the ß-amyrin synthase genes (GmBAS1 and GmBAS2) has proven effective in reducing soyasaponin content in soybean seeds, the specific functional roles of these genes remain unclear. In this study, site-directed mutagenesis was performed on two GmBAS loci using a DNA-free clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system. A complex of sgRNA targeting sequences conserved in the two loci and Cas9 protein was introduced into the shoot apical meristems of soybean embryonic axes via bombardment. Cleaved amplified polymorphic sequences (CAPS) analysis conducted 1 month post-bombardment revealed that 138 seedlings out of 1,467 screened exhibited mutations at one or both GmBAS loci. CAPS and sequencing analysis in the subsequent generation identified a total of 16 plants with inheritable mutations ranging from one to ten nucleotides. High-performance liquid chromatography (HPLC) analysis showed that site-directed mutagenesis in the GmBAS1 locus resulted in the absence of soyasaponins in mature seeds, as well as in young roots, stems, and leaves. These findings demonstrate that GmBAS1 is the predominant ß-amyrin synthase gene in soybean plants. In addition, the DNA-free CRISPR/Cas9 system was shown to be highly efficient in inducing simultaneous mutagenesis at two target loci using a single gRNA.}, }
@article {pmid39780710, year = {2025}, author = {Elkhadragy, L and Carlino, MJ and Jordan, LR and Pennix, T and Ismail, N and Guzman, G and Samuelson, JP and Schook, LB and Schachtschneider, KM and Gaba, RC}, title = {Development of a genetically tailored implantation hepatocellular carcinoma model in Oncopigs by somatic cell CRISPR editing.}, journal = {Disease models & mechanisms}, volume = {18}, number = {1}, pages = {}, doi = {10.1242/dmm.052079}, pmid = {39780710}, issn = {1754-8411}, support = {1R21CA219461//NIH NCI/ ; 1R21CA219461/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Carcinoma, Hepatocellular/genetics/pathology ; *Gene Editing ; *Liver Neoplasms/genetics/pathology ; *Disease Models, Animal ; *PTEN Phosphohydrolase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation/genetics ; Animals, Genetically Modified ; Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; Cell Proliferation ; Cell Movement/genetics ; Cell Line, Tumor ; Swine ; Tumor Suppressor Protein p53/genetics/metabolism ; }, abstract = {Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis, necessitating preclinical models for evaluating novel therapies. Large-animal models are particularly valuable for assessing locoregional therapies, which are widely employed across HCC stages. This study aimed to develop a large-animal HCC model with tailored tumor mutations. The Oncopig, a genetically engineered pig with inducible TP53R167H and KRASG12D, was used in the study. Hepatocytes were isolated from Oncopigs and exposed to Cre recombinase in vitro to create HCC cells, and additional mutations were introduced by CRISPR/Cas9 knockout of PTEN and CDKN2A. These edits increased Oncopig HCC cell proliferation and migration. Autologous HCC cells with these CRISPR edits were implanted into Oncopigs using two approaches: ultrasound-guided percutaneous liver injections, which resulted in the development of localized intrahepatic masses, and portal vein injections, which led to multifocal tumors that regressed over time. Tumors developed by both approaches harbored PTEN and CDKN2A knockout mutations. This study demonstrates the feasibility of developing genetically tailored HCC tumors in Oncopigs using somatic cell CRISPR editing and autologous implantation, providing a valuable large-animal model for in vivo therapeutic assessment.}, }
@article {pmid39641224, year = {2025}, author = {Yu, J and Li, S and Xiong, B and Shen, Y and Guan, X and Zhu, Y and Fang, Y and Zhang, S and Ding, S and Liu, C and Yue, W and Yin, H and Xu, H}, title = {Probiotics Bi-Enzymatic Cascade Repair System for Editing the Inflammatory Microenvironment to Boost Probiotic Therapy in Inflammatory Bowel Disease.}, journal = {Advanced materials (Deerfield Beach, Fla.)}, volume = {37}, number = {4}, pages = {e2412429}, doi = {10.1002/adma.202412429}, pmid = {39641224}, issn = {1521-4095}, support = {82151318//National Natural Science Foundation of China/ ; 82430064//National Natural Science Foundation of China/ ; 82302206//National Natural Science Foundation of China/ ; 82102050//National Natural Science Foundation of China/ ; 82402268//National Natural Science Foundation of China/ ; 82302197//National Natural Science Foundation of China/ ; 82102048//National Natural Science Foundation of China/ ; 2022ZSQD07//Scientific Research and Development Fund of Zhongshan Hospital of Fudan University/ ; 2019LJ21//Shanghai Municipal Health Commission/ ; SHSLCZDZK03502//Shanghai Municipal Health Commission/ ; 23YF1441600//Shanghai Sailing Program/ ; 2023TQ0073//China Postdoctoral Science Foundation/ ; }, mesh = {*Probiotics/therapeutic use ; Animals ; *Inflammatory Bowel Diseases/therapy/metabolism ; Mice ; CRISPR-Cas Systems/genetics ; Gene Editing ; Lacticaseibacillus rhamnosus ; Inflammation/metabolism ; Reactive Oxygen Species/metabolism ; Humans ; Gastrointestinal Microbiome ; }, abstract = {Inflammatory bowel disease presents significant treatment challenges owing to its complex pathology. Although probiotics have shown promise as a therapeutic option, their effectiveness is often limited by low concentrations at sites of inflammation, exacerbated by excessive reactive oxygen species and inflammatory triggers. To address this, an innovative cascade repair system is developed to enhance probiotic therapeutic impact by modulating the intestinal microenvironment. This system uses iMXene's catalytic properties to neutralize reactive oxygen species in the gut and its capacity to deliver the CRISPR/dCas9 gene editing system to activate the NLR family pyrin domain containing 12 genes, helping suppress inflammation. By promoting the colonization of Lactobacillus rhamnosus, the system inhibits inflammation pathways and supports the restoration of a balanced intestinal flora through a cascade repair mechanism. These findings demonstrate significant therapeutic benefits in experimental models, with improvements in the overall well-being of treated mice and effective repair of intestinal inflammation damage. This pioneering approach holds promise for inflammatory bowel disease treatment and opens new avenues for managing other inflammatory conditions, offering valuable insights and guidance for future research into inflammatory diseases.}, }
@article {pmid39630114, year = {2025}, author = {Guan, X and Yang, R and Zhang, J and Moon, J and Hou, C and Guo, C and Avery, L and Scarola, D and Roberts, DS and LaSala, R and Liu, C}, title = {Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {12}, number = {4}, pages = {e2411021}, doi = {10.1002/advs.202411021}, pmid = {39630114}, issn = {2198-3844}, support = {U01CA269147/NH/NIH HHS/United States ; U01AI148306/NH/NIH HHS/United States ; U01CA269147/NH/NIH HHS/United States ; U01AI148306/NH/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Sensitivity and Specificity ; Papillomaviridae/genetics ; Papillomavirus Infections/diagnosis/genetics/virology ; Nucleic Acids/genetics ; Human papillomavirus 16/genetics ; Female ; Uterine Cervical Neoplasms/genetics/diagnosis ; }, abstract = {CRISPR-Cas12a works like a sophisticated algorithm in nucleic acid detection, yet its challenge lies in sometimes failing to distinguish targets with mismatches due to its specificity limitations. Here, the mismatch profiles, including the quantity, location, and type of mismatches in the CRISPR-Cas12a reaction, are investigated and its various tolerances to mismatches are discovered. By harnessing the specificity defect of the CRISPR-Cas12a enzyme, a dual-mode detection strategy is designed, which includes approximate matching and precise querying of target sequences and develop a programmable multiplexed nucleic acid assay. With the assay, 14 high-risk human papillomavirus (HPV) subtypes are simultaneously detected, collectively responsible for 99% of cervical cancer cases, with attomolar sensitivity. Specifically, the assay not only distinguishes HPV16 and HPV18, the two most common subtypes but also detects 12 other high-risk pooled HPV subtypes. To enable low-cost point-of-care testing, the assay is incorporated into a paper-based microfluidic chip. Furthermore, the clinical performance of the paper-based microfluidic chip is validated by testing 75 clinical swab samples, achieving performance comparable to that of PCR. This programmable multiplexed nucleic acid assay has the potential to be widely applied for sensitive, specific, and simultaneous detection of different pathogens.}, }
@article {pmid39622167, year = {2025}, author = {Maity, A and Sathyanarayanan, A and Kumar, R and Vora, J and Gawde, J and Jain, H and Bagal, B and Subramanian, PG and Sengar, M and Khattry, N and Patkar, N and Hasan, SK}, title = {RAPID-CRISPR: highly sensitive diagnostic assay for detection of PML::RARA isoforms in acute promyelocytic leukemia.}, journal = {Blood advances}, volume = {9}, number = {3}, pages = {463-472}, doi = {10.1182/bloodadvances.2024014539}, pmid = {39622167}, issn = {2473-9537}, mesh = {Humans ; *Leukemia, Promyelocytic, Acute/diagnosis/genetics ; *Oncogene Proteins, Fusion/genetics ; Male ; Female ; Protein Isoforms ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Adult ; Middle Aged ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Retinoic Acid Receptor alpha/genetics ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Acute promyelocytic leukemia (APL), distinguished by the presence of PML::RARA fusion transcript, is a medical emergency because of its high early death rate, which is preventable when diagnosed early. Current diagnostic methods are precise and reliable but are time intensive, require sophisticated instruments, and analytical expertise. This study has redefined APL identification by CRISPR system (RAPID-CRISPR) to rapidly (<3 hours) detect PML::RARA. APL cell lines (NB4 and UF-1) and bone marrow/peripheral blood samples from 74 patients with APL (66/8, retrospective/prospective) and 48 controls were included in the study. We used a DETECTR (DNA endonuclease-targeted CRISPR transreporter) assay to identify the bcr1, bcr2, and bcr3 PML::RARA isoforms. To ensure high specificity, we used PML::RARA-specific loop-mediated isothermal amplification (LAMP) primers, synthetic protospacer-adjacent motif sites, and isoform-specific CRISPR RNAs. RAPID-CRISPR recognized APL with 100% sensitivity and 100% specificity in an ambispective cohort of patient samples. Furthermore, our blinded validation approach to detect PML::RARA in an unbiased manner provides an additional layer in the diagnostic precision of APL. RAPID-CRISPR demonstrated superior sensitivity, detecting as few as 1 copy of PML::RARA compared with 10 copies by the gold-standard reverse transcriptase qualitative and quantitative polymerase chain reaction. The nucleic acid extraction-free protocol combined with the 1-step reverse transcriptase LAMP-based DETECTR followed by lateral flow readout makes the RAPID-CRISPR assay suitable for diagnosing APL in point-of-care settings. This simple, cost-effective tool, with its easy-to-read format, is particularly valuable in underresourced regions. The assay facilitates timely diagnosis and prompt administration of lifesaving therapies such as all-trans retinoic acid and arsenic trioxide in APL.}, }
@article {pmid39445704, year = {2025}, author = {Cohn, DM and Gurugama, P and Magerl, M and Katelaris, CH and Launay, D and Bouillet, L and Petersen, RS and Lindsay, K and Aygören-Pürsün, E and Maag, D and Butler, JS and Shah, MY and Golden, A and Xu, Y and Abdelhady, AM and Lebwohl, D and Longhurst, HJ}, title = {CRISPR-Based Therapy for Hereditary Angioedema.}, journal = {The New England journal of medicine}, volume = {392}, number = {5}, pages = {458-467}, doi = {10.1056/NEJMoa2405734}, pmid = {39445704}, issn = {1533-4406}, mesh = {Humans ; Male ; Female ; Adult ; *Angioedemas, Hereditary/drug therapy/genetics/therapy ; Middle Aged ; Double-Blind Method ; Genetic Therapy/adverse effects ; Gene Editing ; Plasma Kallikrein/antagonists & inhibitors ; Kallikreins/blood ; CRISPR-Cas Systems ; Young Adult ; Aged ; }, abstract = {BACKGROUND: Hereditary angioedema is a rare genetic disease characterized by severe and unpredictable swelling attacks. NTLA-2002 is an in vivo gene-editing therapy that is based on clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9. NTLA-2002 targets the gene encoding kallikrein B1 (KLKB1). A single dose of NTLA-2002 may provide lifelong control of angioedema attacks.
METHODS: In this phase 2 portion of a phase 1-2 trial, we randomly assigned adults with hereditary angioedema in a 2:2:1 ratio to receive NTLA-2002 in a single dose of 25 mg or 50 mg or placebo. The primary end point was the number of angioedema attacks per month (the monthly attack rate) from week 1 through week 16. Secondary end points included safety, pharmacokinetics, and pharmacodynamics (i.e., the change from baseline in total plasma kallikrein protein level); exploratory end points included patient-reported outcomes.
RESULTS: Of the 27 patients who underwent randomization, 10 received 25 mg of NTLA-2002, 11 received 50 mg, and 6 received placebo. From week 1 through week 16, the estimated mean monthly attack rate was 0.70 (95% confidence interval [CI], 0.25 to 1.98) with 25 mg of NTLA-2002, 0.65 (95% CI, 0.24 to 1.76) with 50 mg, and 2.82 (95% CI, 0.80 to 9.89) with placebo; the difference in the estimated mean attack rate with NTLA-2002 as compared with placebo was -75% with 25 mg and -77% with 50 mg. Among patients who received NTLA-2002, 4 of the 10 patients who received 25 mg (40%) and 8 of the 11 who received 50 mg (73%) were attack-free with no additional treatment during the period from week 1 through week 16. The most common adverse events among patients who received NTLA-2002 were headache, fatigue, and nasopharyngitis. The mean percent change in total plasma kallikrein protein levels from baseline to week 16 was -55% with 25 mg and -86% with 50 mg; levels remained unchanged with placebo.
CONCLUSIONS: NTLA-2002 administered in a single dose of 25 mg or 50 mg reduced angioedema attacks and led to robust and sustained reduction in total plasma kallikrein levels in patients with hereditary angioedema. These results support continued investigation in a larger phase 3 trial. (Funded by Intellia Therapeutics; ClinicalTrials.gov number, NCT05120830; EudraCT number, 2021-001693-33.).}, }
@article {pmid39264585, year = {2025}, author = {Lou, H and Wang, X and Jiang, Q and Li, X and Yao, Y and Chen, Q and Chen, L and Zhang, S and Yu, Y and Liu, C and Zhou, H}, title = {Clinical evaluation of a highly multiplexed CRISPR-based diagnostic assay for diagnosing lower respiratory tract infection: a prospective cohort study.}, journal = {Infectious diseases (London, England)}, volume = {57}, number = {2}, pages = {167-177}, doi = {10.1080/23744235.2024.2402921}, pmid = {39264585}, issn = {2374-4243}, mesh = {Humans ; Prospective Studies ; Male ; Female ; Middle Aged ; *Respiratory Tract Infections/diagnosis/microbiology ; Aged ; *Sensitivity and Specificity ; Adult ; *Multiplex Polymerase Chain Reaction/methods ; High-Throughput Nucleotide Sequencing/methods ; Molecular Diagnostic Techniques/methods ; Bacteria/genetics/isolation & purification/classification ; Young Adult ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Aged, 80 and over ; CRISPR-Cas Systems ; Bacterial Infections/diagnosis/microbiology ; Adolescent ; }, abstract = {OBJECTIVE: Accurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a.
METHODS: We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology.
RESULTS: The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%).
CONCLUSIONS: Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected.}, }
@article {pmid39871009, year = {2025}, author = {Sakthivel, K and Balasubramanian, R and Sampathrajan, V and Veerasamy, R and Appachi, SV and K K, K}, title = {Transforming tomatoes into GABA-rich functional foods through genome editing: A modern biotechnological approach.}, journal = {Functional & integrative genomics}, volume = {25}, number = {1}, pages = {27}, pmid = {39871009}, issn = {1438-7948}, mesh = {*Gene Editing/methods ; *gamma-Aminobutyric Acid/metabolism ; *Solanum lycopersicum/genetics/metabolism ; *CRISPR-Cas Systems ; Functional Food ; Plant Breeding/methods ; Humans ; Genome, Plant ; }, abstract = {Gamma-aminobutyric acid (GABA) functions as an inhibitory neurotransmitter which blocks the impulses between nerve cells in the brain. Due to the increasing awareness about the health promoting benefits associated with GABA, it is also artificially synthesized and consumed as a nutritional supplement by people in some regions of the world. Though among the fresh vegetables, tomato fruits do contain a comparatively higher amount of GABA (0.07 to 2.01 mg g[-1] FW), it needs to be further enhanced to fully impart its potential health benefits. Achieving this feat through classical breeding approaches is time and resource consuming, and is also associated with linkage drag. On the other hand, precise targeting of specific sites in the genome with less off- target effects is mediated by CRISPR/Cas9 genome editing tool and is widely used to overcome the barriers associated with traditional breeding approaches. Combining genome editing with speed breeding techniques can enable the rapid development of GABA-rich tomato cultivars, paving a way to unlock a new era of functional foods, where every bite contributes to cognitive well-being and holistic health. This review highlights the significance of GABA boosted functional foods and explores the potential of CRISPR/Cas9 technology for developing GABA enriched tomatoes.}, }
@article {pmid39870618, year = {2025}, author = {Ray-Jones, H and Sung, CK and Chan, LT and Haglund, A and Artemov, P and Della Rosa, M and Ruje, L and Burden, F and Kreuzhuber, R and Litovskikh, A and Weyenbergh, E and Brusselaers, Z and Tan, VXH and Frontini, M and Wallace, C and Malysheva, V and Bottolo, L and Vigorito, E and Spivakov, M}, title = {Genetic coupling of enhancer activity and connectivity in gene expression control.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {970}, pmid = {39870618}, issn = {2041-1723}, support = {MC-A652-5QA20//RCUK | Medical Research Council (MRC)/ ; MC_UU_00002/4//RCUK | Medical Research Council (MRC)/ ; MR/W029790/1//RCUK | Medical Research Council (MRC)/ ; MC_UU_00002/4//RCUK | Medical Research Council (MRC)/ ; FS/18/53/33863//British Heart Foundation (BHF)/ ; RE/18/1/34212//British Heart Foundation (BHF)/ ; WT220788//Wellcome Trust (Wellcome)/ ; EP/N510129/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; }, mesh = {*Enhancer Elements, Genetic ; *Gene Expression Regulation ; Humans ; Quantitative Trait Loci/genetics ; Monocytes/metabolism ; Cells, Cultured ; Chromatin/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Transcription Factors/metabolism ; CCCTC-Binding Factor/metabolism ; Protein Binding ; Base Sequence ; Male ; }, abstract = {Gene enhancers often form long-range contacts with promoters, but it remains unclear if the activity of enhancers and their chromosomal contacts are mediated by the same DNA sequences and recruited factors. Here, we study the effects of expression quantitative trait loci (eQTLs) on enhancer activity and promoter contacts in primary monocytes isolated from 34 male individuals. Using eQTL-Capture Hi-C and a Bayesian approach considering both intra- and inter-individual variation, we initially detect 19 eQTLs associated with enhancer-eGene promoter contacts, most of which also associate with enhancer accessibility and activity. Capitalising on these shared effects, we devise a multi-modality Bayesian strategy, identifying 629 "trimodal QTLs" jointly associated with enhancer accessibility, eGene promoter contact, and gene expression. Causal mediation analysis and CRISPR interference reveal causal relationships between these three modalities. Many detected QTLs overlap disease susceptibility loci and influence the predicted binding of myeloid transcription factors, including SPI1, GABPB and STAT3. Additionally, a variant associated with PCK2 promoter contact directly disrupts a CTCF binding motif and impacts promoter insulation from downstream enhancers. Jointly, our findings suggest an inherent genetic coupling of enhancer activity and connectivity in gene expression control relevant to human disease and highlight the regulatory role of genetically determined chromatin boundaries.}, }
@article {pmid39870449, year = {2025}, author = {Ai, X and Tang, Z}, title = {Aptazyme-directed A-to-I RNA editing.}, journal = {Methods in enzymology}, volume = {710}, number = {}, pages = {267-283}, doi = {10.1016/bs.mie.2024.11.022}, pmid = {39870449}, issn = {1557-7988}, mesh = {*RNA Editing ; Humans ; *Adenosine Deaminase/metabolism/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Inosine/genetics/metabolism ; Adenosine/metabolism/genetics ; HEK293 Cells ; Aptamers, Nucleotide/genetics ; }, abstract = {As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.}, }
@article {pmid39870447, year = {2025}, author = {Bhakta, S and Tsukahara, T}, title = {Restoration of G to A mutated transcripts using the MS2-ADAR1 system.}, journal = {Methods in enzymology}, volume = {710}, number = {}, pages = {229-240}, doi = {10.1016/bs.mie.2024.11.031}, pmid = {39870447}, issn = {1557-7988}, mesh = {*Adenosine Deaminase/genetics/metabolism ; Humans ; *RNA-Binding Proteins/genetics/metabolism ; RNA Editing ; Guanosine/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; Levivirus/genetics ; RNA, Messenger/genetics ; Point Mutation ; Mutation ; }, abstract = {Site-directed RNA editing (SDRE) holds significant promise for treating genetic disorders resulting from point mutations. Gene therapy, for common genetic illnesses is becoming more popular and, although viable treatments for genetic disorders are scarce, stop codon mutation-related conditions may benefit from gene editing. Effective SDRE generally depends on introducing many guideRNA molecules relative to the target gene; however, large ratios cannot be achieved in the context of gene therapy applications. Gene-encoded information can be altered, and functionally diverse proteins produced from a single gene by restoration of point-mutated RNA molecules using SDRE. Adenosine deaminase acting on RNA (ADAR) is an RNA-editing enzyme, that can specifically convert adenosine (A) residues to inosines (I), which are translated as guanosine (G). MS2 system along with ADAR1 deaminase domain can target a particular A and repair G to A mutations. In this study, we used the RNA binding MS2 coat protein fused with the ADAR1 deaminase domain controlled by the CMV promoter, and a 19 bp guide RNA (complementary to the target mRNA sequence) engineered with 6 × MS2 stem-loops downstream or 1 × MS2 stem-loop (double MS2) on either side, controlled by the U6 promoter. When the EGFP TGG codon (tryptophan) was altered to an amber (TAG), opal (TGA), or ochre (TAA) stop codon, the modified ADAR1 deaminase domain could convert A-to-I (G) at the edited sites. It is anticipated that successful establishment of this technique will result in a new era in gene therapy, allowing remarkably efficient gene repair, even in vivo.}, }
@article {pmid39823408, year = {2025}, author = {Robert, NM and Ferrier-Tarin, S and Tremblay, JJ}, title = {A New Leydig Cell-Exclusive Cre Line Allows Lineage Tracing of Fetal and Adult Leydig Cell Populations in the Mouse.}, journal = {Endocrinology}, volume = {166}, number = {2}, pages = {}, doi = {10.1210/endocr/bqaf012}, pmid = {39823408}, issn = {1945-7170}, support = {MOP-81387/CAPMC/CIHR/Canada ; //Fondation du CHU de Québec-Université Laval/ ; }, mesh = {Animals ; *Leydig Cells/metabolism/cytology ; Male ; Mice ; *Integrases/genetics/metabolism ; *Cell Lineage ; *Insulin/metabolism ; *Proteins/genetics/metabolism ; Fetus/metabolism/cytology ; Mice, Transgenic ; Testis/metabolism/cytology/embryology ; CRISPR-Cas Systems ; Female ; }, abstract = {Leydig cells produce hormones that are required for male development, fertility, and health. Two Leydig cell populations produce these hormones but at different times during development: fetal Leydig cells, which are active during fetal life, and adult Leydig cells, which are functional postnatally. Historically, our ability to understand the origin and function of Leydig cells has been made difficult by the lack of genetic models to exclusively target these cells. Taking advantage of the Leydig cell-exclusive expression pattern of the Insl3 gene, we used a CRISPR/Cas9 gene-editing strategy to knock-in iCre recombinase into the mouse Insl3 locus. To demonstrate the Leydig cell-exclusive nature of our iCre line, lineage-tracing experiments were performed by crossing Insl3iCre mice with a Rosa26LoxSTOPLox-TdTomato reporter. iCre activity was restricted to male offspring. TdTomato fluorescence was detected both in fetal and adult Leydig cells and colocalized with CYP17A1, a classic Leydig cell marker. Prior to birth, fluorescence was observed in fetal Leydig cells beginning at embryonic day 13.0. Fluorescence was also detected in adult Leydig cells starting at postnatal day 5 and continuing to the mature testis. Fluorescence was not detected in any other fetal or adult tissue examined, except for the unexpected finding that the adrenal cortex contains some Insl3-expressing Leydig-like cells. Our Leydig cell-exclusive iCre line therefore constitutes an invaluable new tool to study not only the origin of Leydig cells but also to target genes that have been long-proposed to be important for the development and functioning of these critical endocrine cells.}, }
@article {pmid39814565, year = {2025}, author = {Chau, CCC and Weckman, NE and Thomson, EE and Actis, P}, title = {Solid-State Nanopore Real-Time Assay for Monitoring Cas9 Endonuclease Reactivity.}, journal = {ACS nano}, volume = {19}, number = {3}, pages = {3839-3851}, doi = {10.1021/acsnano.4c15173}, pmid = {39814565}, issn = {1936-086X}, mesh = {*Nanopores ; *CRISPR-Cas Systems/genetics ; DNA/chemistry/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Endonucleases/metabolism/chemistry ; }, abstract = {The field of nanopore sensing is now moving beyond nucleic acid sequencing. An exciting avenue is the use of nanopore platforms for the monitoring of biochemical reactions. Biological nanopores have been used for this application, but solid-state nanopore approaches have lagged. This is due to the necessity of using higher salt conditions (e.g., 4 M LiCl) to improve the signal-to-noise ratio which completely abolish the activities of many biochemical reactions. We pioneered a polymer electrolyte solid-state nanopore approach that maintains a high signal-to-noise ratio even at a physiologically relevant salt concentration. Here, we report the monitoring of the restriction enzyme SwaI and CRISPR-Cas9 endonuclease activities under physiological salt conditions and in real time. We investigated the dsDNA cleavage activity of these enzymes in a range of digestion buffers and elucidated the off-target activity of CRISPR-Cas9 ribonucleoprotein endonuclease in the presence of single base pair mismatches. This approach enables the application of solid-state nanopores for the dynamic monitoring of biochemical reactions under physiological salt conditions.}, }
@article {pmid39740438, year = {2025}, author = {Shen, Y and Li, B and Hao, G and Duan, M and Zhao, Y and Liu, Z and Li, X and Jia, F}, title = {A CRISPR/Cas12a-based direct transverse relaxation time biosensor via hydrogel sol-gel transition for Salmonella detection.}, journal = {Food chemistry}, volume = {470}, number = {}, pages = {142693}, doi = {10.1016/j.foodchem.2024.142693}, pmid = {39740438}, issn = {1873-7072}, mesh = {*Biosensing Techniques/instrumentation ; *Salmonella/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Hydrogels/chemistry ; Bacterial Proteins/genetics/metabolism/chemistry ; Phase Transition ; }, abstract = {This research developed a magnetic relaxation switching (MRS) biosensor based on hydrogel sol-gel transition and the CRISPR/Cas12a system (MRS-CRISPR) to detect Salmonella. Herein, the alkaline phosphatase (ALP) labeled with streptavidin was captured by the biotin-modified DNA on magnetic nanoparticles (MNPs) surface, which generated an acidic environment via enzymatic reaction to release Ca[2+] and induced the transformation of alginate sol to hydrogels. In contrast, Salmonella activated the trans-cleavage activity of the CRISPR/Cas12a system, interrupting the capture of ALP and the subsequent sol-gel transition. Then, transverse relaxation time (T2), which was regulated by the hydrogelation process was measured for Salmonella detection. The MRS-CRISPR biosensor enables sensitive detection of Salmonella with a detection limit of 158 CFU/mL. It directly alters the state of water molecules, overcoming the disadvantages of traditional MRS sensors that rely on MNPs to produce T2 signals indirectly. This method offers innovative insights for the application of the MRS technology in food safety analysis.}, }
@article {pmid39688405, year = {2025}, author = {Allan-Blitz, L-T and Adams, G and Sanders, G and Shah, P and Ramesh, K and Jarolimova, J and Ard, KL and Branda, JA and Klausner, JD and Sabeti, PC and Lemieux, JE}, title = {Preliminary clinical performance of a Cas13a-based lateral flow assay for detecting Neisseria gonorrhoeae in urine specimens.}, journal = {mSphere}, volume = {10}, number = {1}, pages = {e0067724}, doi = {10.1128/msphere.00677-24}, pmid = {39688405}, issn = {2379-5042}, support = {U19AI110818//National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2019123//Doris Duke Charitable Foundation (DDCF)/ ; 2021287//Doris Duke Charitable Foundation (DDCF)/ ; //American Sexually Transmitted Diseases Association (ASTDA)/ ; K23AI182453//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {Humans ; *Neisseria gonorrhoeae/genetics/isolation & purification ; *Gonorrhea/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics/urine ; CRISPR-Cas Systems ; Machine Learning ; Molecular Diagnostic Techniques/methods ; Urethritis/microbiology/diagnosis/urine ; Point-of-Care Testing ; Sensitivity and Specificity ; Smartphone ; Point-of-Care Systems ; }, abstract = {Nucleic acid amplification testing (NAAT) for N. gonorrhoeae is unavailable in resource-limited settings. We previously developed a CRISPR-based lateral flow assay for detecting N. gonorrhoeae. We aimed to pair that assay with point-of-care DNA extraction, assess performance in clinical urine specimens, and optimize assay kinetics. We collected urine specimens among men presenting with urethritis enrolling in a clinical trial at the Massachusetts General Hospital Sexual Health Clinic. We assessed the quantified DNA yield of detergent-based extractions with and without heat. We selected one detergent for extracting all specimens, paired with isothermal recombinase polymerase amplification for 90 minutes and lateral flow Cas13a detection, interpreted via pixel intensity analysis. We also trained a smartphone-based machine-learning model on 1,008 images to classify lateral flow results. We used the model to interpret lateral flow results from the clinical specimens. We also tested a modified amplification chemistry with a second forward primer lacking the T7-promoter to accelerate reaction kinetics. Extraction with 0.02% Triton X resulted in an average DNA yield of 2.6 × 10[6] copies/µL (SD ± 6.7 × 10[5]). We treated 40 urine specimens (n = 12 positive) with 0.02% Triton X, and using quantified pixel intensity analysis, the Cas13a-based assay correctly classified all specimens (100% agreement; 95% CI 91.2%-100%). The machine-learning model correctly classified 45/45 strips in the validation data set and all 40 lateral flow strips from clinical specimens. Including the second forward primer reduced incubation time to 60 minutes. Using point-of-care DNA extraction, our Cas13a-based lateral flow N. gonorrhoeae assay demonstrated promising performance among clinical urine specimens.IMPORTANCEUsing a CRISPR-based assay we previously developed for Neisseria gonorrhoeae detection, we developed new techniques to facilitate point-of-care use. We then demonstrated the promising performance of that assay in clinical specimens. Furthermore, we developed a smartphone-based machine learning application for assisting interpretation of lateral flow strip results. Such an assay has the potential to transform the care of sexually transmitted infections in low-resource settings where diagnostic tests are unavailable. A point-of-care pathogen-specific assay, paired with the connectivity offered by a smartphone application, can also support public health surveillance efforts in such areas.}, }
@article {pmid39687993, year = {2025}, author = {Chen, S and Pinto Carneiro, S and Merkel, OM}, title = {Anionic polymer coating for enhanced delivery of Cas9 mRNA and sgRNA nanoplexes.}, journal = {Biomaterials science}, volume = {13}, number = {3}, pages = {659-676}, pmid = {39687993}, issn = {2047-4849}, mesh = {Humans ; *RNA, Messenger/administration & dosage/chemistry ; A549 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Polyethylene Glycols/chemistry ; Proto-Oncogene Proteins p21(ras)/genetics ; Gene Editing ; Anions/chemistry ; CRISPR-Cas Systems ; Polymers/chemistry ; }, abstract = {Polymeric carriers have long been recognized as some of the most effective and promising systems for nucleic acid delivery. In this study, we utilized an anionic di-block co-polymer, PEG-PLE, to enhance the performance of lipid-modified PEI (C14-PEI) nanoplexes for delivering Cas9 mRNA and sgRNA targeting KRAS G12S mutations in lung cancer cells. Our results demonstrated that PEG-PLE, when combined with C14-PEI at a weight-to-weight ratio of 0.2, produced nanoplexes with a size of approximately 140 nm, a polydispersity index (PDI) of 0.08, and a zeta potential of around -1 mV. The PEG-PLE/C14-PEI nanoplexes at this ratio were observed to be both non-cytotoxic and effective in encapsulating Cas9 mRNA and sgRNA. Confocal microscopy imaging revealed efficient endosomal escape and intracellular distribution of the RNAs. Uptake pathway inhibition studies indicated that the internalization of PEG-PLE/C14-PEI primarily involves scavenger receptors and clathrin-mediated endocytosis. Compared to C14-PEI formulations, PEG-PLE/C14-PEI demonstrated a significant increase in luciferase mRNA expression and gene editing efficiency, as confirmed by T7EI and ddPCR, in A549 cells. Sanger sequencing identified insertions and/or deletions around the PAM sequence, with a total of 69% indels observed. Post-transfection, the KRAS-ERK pathway was downregulated, resulting in significant increases in cell apoptosis and inhibition of cell migration. Taken together, this study reveals a new and promising formulation for CRISPR delivery as potential lung cancer treatment.}, }
@article {pmid39637570, year = {2025}, author = {Ding, L and Cao, S and Bai, L and He, S and He, L and Wang, Y and Wu, Y and Yu, S}, title = {Versatile fluorescence biosensors based on CRISPR/Cas12a for determination of site-specific DNA methylation from blood and tissues.}, journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy}, volume = {329}, number = {}, pages = {125520}, doi = {10.1016/j.saa.2024.125520}, pmid = {39637570}, issn = {1873-3557}, mesh = {*DNA Methylation ; Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Spectrometry, Fluorescence/methods ; DNA/genetics ; }, abstract = {The identification of DNA methylation at specific sites is crucial for the early detection of cancer since DNA methylation is intimately associated to the occurrence and development of cancer. Herein, two types of sensors that can detect site-specific DNA methylation were developed to meet practical requirements using methylation sensitive restriction endonuclease and CRISPR/Cas12a. To accomplish rapid detection of target, an AciI-mediated CRISPR/Cas12a assay was developed by coupling AciI to recognize DNA methylation with Cas12a to identify site-specific DNA. Since protospacer adjacent motif (PAM)-dependent endonuclease activity and trans-cleavage activity of Cas12a, it is possible to detect site-specific DNA methylation within 2 h with high specificity and acceptable sensitivity. To satisfy the needs of trace target detection, we developed an GlaI-strand displacement amplification (SDA) assisted CRISPR/Cas12a system. The system converts double-stranded methylated DNA to abundant single-stranded by GlaI and SDA. Then, the combination of SDA and CRISPR/Cas12a enable cascades amplification of signal. The approach can therefore be used to detect methylation at different specified sites, even those without PAM, and can increase sensitivity with a detection limit down to 8.19 fM. Importantly, the assay can distinguish between colorectal cancer and precancerous tissue, as well as identify colorectal patients and healthy people. This study provides a new avenue for the development of new biosensors for methylation analysis, and the two methods devised have the potential to meet the multiple requirements of site-specific methylation testing in various clinical settings.}, }
@article {pmid39872948, year = {2023}, author = {Sun, Y and Lin, W and Kaundal, R and Chi, T}, title = {iMAPping the Perturb-Atlas.}, journal = {Life medicine}, volume = {2}, number = {1}, pages = {lnac057}, pmid = {39872948}, issn = {2755-1733}, abstract = {A key objective of the research in the postgenomic era is to decipher the functions of the mammalian genome, which has remained largely enigmatic despite intensive efforts in the functional genomics field over the past two decades. To attack this problem, we have combined the CRISPR-Cas and Cre-Lox technologies to develop iMAP (inducible Mosaic Animal for Perturbation), a transformative tool for rapidly unraveling mammalian genome function in situ. Furthermore, we have used iMAP to rapidly construct a "Perturb-Atlas" profiling the functions of 90 protein-coding genes across 39 tissues in mice, which has offered rich insights into gene functions difficult to readily obtain using conventional mouse gene-targeting models. In this research highlight, we offer a brief primer on the iMAP technology, outlining its mechanism, strengths and limitations, and pointing out future directions of research in the area.}, }
@article {pmid39870445, year = {2025}, author = {Campbell, KB and Cheng, J and Mendoza, HG and Karki, A and Beal, PA and Fisher, AJ}, title = {Structural analysis of human ADAR2-RNA complexes by X-ray crystallography.}, journal = {Methods in enzymology}, volume = {710}, number = {}, pages = {19-53}, doi = {10.1016/bs.mie.2024.11.023}, pmid = {39870445}, issn = {1557-7988}, mesh = {*Adenosine Deaminase/chemistry/metabolism ; Humans ; *RNA-Binding Proteins/chemistry/metabolism ; Crystallography, X-Ray/methods ; RNA Editing ; Protein Binding ; RNA/chemistry/metabolism ; RNA, Guide, CRISPR-Cas Systems/chemistry ; }, abstract = {Adenosine deaminases acting on RNAs (ADARs) are a class of RNA editing enzymes found in metazoa that catalyze the hydrolytic deamination of adenosine to inosine in duplexed RNA. Inosine is a nucleotide that can base pair with cytidine, therefore, inosine is interpreted by cellular processes as guanosine. ADARs are functionally important in RNA recoding events, RNA structure modulation, innate immunity, and can be harnessed for therapeutically-driven base editing to treat genetic disorders. Guide RNAs (gRNAs) bearing various modifications can be used to recruit ADARs to edit sites of interest in a process called site-directed RNA editing (SDRE). To help advance the rational design of gRNAs for therapeutics, characterizing the structure-to-activity relationship of ADARs' recognition and binding of substrate duplex RNA at atomic resolution is critical. In this chapter, we describe the process of determining the structure of human ADAR2 bound to duplex RNA using X-ray crystallography. Solid phase synthesis of 8-azanebularine-modified RNAs and purification for binding and crystallographic studies are described. The overexpression and purification of ADARs and assembly of the protein-RNA complex are detailed. Lastly, methods for crystallizing ADAR-RNA complexes and X-ray structure determination and data refinement strategies are outlined.}, }
@article {pmid39870442, year = {2025}, author = {Salvador, PJ and Dugan, NM and Ouye, R and Beal, PA}, title = {En masse evaluation of RNA guides (EMERGe) for ADARs.}, journal = {Methods in enzymology}, volume = {710}, number = {}, pages = {131-152}, doi = {10.1016/bs.mie.2024.11.030}, pmid = {39870442}, issn = {1557-7988}, mesh = {Humans ; *Adenosine Deaminase/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *High-Throughput Nucleotide Sequencing/methods ; RNA Editing ; Adenosine/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Gene Library ; }, abstract = {Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in duplex RNA, and through the delivery of guide RNAs, can be directed to edit specific adenosine sites. As ADARs are endogenously expressed in humans, their editing capacities hold therapeutic potential and allow us to target disease-relevant sequences in RNA through the rationale design of guide RNAs. However, current design principles are not suitable for difficult-to-edit target sites, posing challenges to unlocking the full therapeutic potential of this approach. This chapter discusses how we circumvent this barrier through an in vitro screening method, En Masse Evaluation of RNA Guides (EMERGe), which enables comprehensive screening of ADAR substrate libraries and facilitates the identification of editing-enabling guide strands for specific adenosines. From library generation and screening to next generation sequencing (NGS) data analysis to verification experiments, we describe how a sequence of interest can be identified through this high-throughput screening method. Furthermore, we discuss downstream applications of selected guide sequences, challenges in maximizing library coverage, and potential to couple the screen with machine learning or deep learning models.}, }
@article {pmid39870441, year = {2025}, author = {Akira, A and Levanon, E and Ben Aroya, S}, title = {Leveraging Saccharomyces cerevisiae for ADAR research: From high-yield purification to high-throughput screening and therapeutic applications.}, journal = {Methods in enzymology}, volume = {710}, number = {}, pages = {1-18}, doi = {10.1016/bs.mie.2024.11.026}, pmid = {39870441}, issn = {1557-7988}, mesh = {*Saccharomyces cerevisiae/genetics ; *Adenosine Deaminase/metabolism/genetics/isolation & purification ; *High-Throughput Screening Assays/methods ; *RNA Editing ; *RNA-Binding Proteins/genetics/metabolism/isolation & purification ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Saccharomyces cerevisiae, a model eukaryotic organism with a rich history in research and industry, has become a pivotal tool for studying Adenosine Deaminase Acting on RNA (ADAR) enzymes despite lacking these enzymes endogenously. This chapter reviews the diverse methodologies harnessed using yeast to elucidate ADAR structure and function, emphasizing its role in advancing our understanding of RNA editing. Initially, Saccharomyces cerevisiae was instrumental in the high-yield purification of ADARs, addressing challenges associated with enzyme stability and activity in other systems. The chapter highlights the successful application of yeast in high-throughput screening platforms that identify key structural motifs and substrate preferences of ADARs, showcasing its utility in revealing complex enzyme mechanics. Furthermore, we discuss the development of yeast-based systems to optimize guide RNA sequences for site-directed RNA editing (SDRE), demonstrating how these systems can be employed to refine therapeutic strategies targeting genetic mutations. Additionally, exogenous expression of ADARs from various species in yeast has shed light on enzyme potency and substrate recognition across different temperatures, offering insights into evolutionary adaptations. Overall, Saccharomyces cerevisiae has proven to be an invaluable asset in ADAR research, facilitating significant advances in our understanding of RNA editing mechanisms and therapeutic applications.}, }
@article {pmid39869590, year = {2025}, author = {Brady, DJ and Saviane, A and Battistolli, M and Varponi, I and Barca, F and Shiomi, K and Cappellozza, S and Sandrelli, F}, title = {Enhanced silk production and pupal weight in Bombyx mori through CRISPR/Cas9-mediated circadian Clock gene disruption.}, journal = {PloS one}, volume = {20}, number = {1}, pages = {e0317572}, doi = {10.1371/journal.pone.0317572}, pmid = {39869590}, issn = {1932-6203}, mesh = {*Bombyx/genetics/growth & development ; Animals ; *CRISPR-Cas Systems ; *Pupa/genetics/growth & development ; *Circadian Clocks/genetics ; *Silk/genetics ; Larva/genetics/growth & development ; Insect Proteins/genetics/metabolism ; }, abstract = {The domesticated silkworm, Bombyx mori, is crucial for global silk production, which is a significant economic activity supporting millions of livelihoods worldwide. Beyond traditional silk production, the growing demand for insect larvae in cosmetics, biomedical products, and animal feed underscores the need to enhance B. mori productivity. This study investigates the role of the circadian clock gene Clock in B. mori using CRISPR/Cas9-mediated mutagenesis to establish the ClkΔ29 knock-out mutant strain. Dysregulation of the circadian clock in ClkΔ29 was demonstrated by altered temporal transcriptional profiles of core circadian clock genes in adult heads and disrupted circadian-controlled behaviors, including adult eclosion and egg hatching rhythms under constant darkness. By analysing larval development timing, as well as the weights of late instar larvae, pupae, and cocoon components in ClkΔ29 mutants and in ClkΔ1922 silkworms (carrying an independently generated Clk- null allele), we showed that CLK contributes to physiological processes regulating B. mori development and growth. Importantly, ClkΔ29 mutants reared on a standard sericulture diet exhibited significant increases in key economic traits, with silk production increasing by up to 7%, and pupal weight increasing by up to 25% compared to wild-type controls. This study highlights the potential of circadian clock gene manipulation to significantly enhance sericultural productivity. Future research should focus on elucidating the molecular mechanisms driving these phenotypes and determining whether they result from circadian clock functions or pleiotropic effects of B. mori Clk. These findings provide a foundation for advancing sustainable sericulture and developing new commercial applications for silkworm-derived products.}, }
@article {pmid39869587, year = {2025}, author = {Kumari, Y and Gunathilaka, N and Amarasinghe, D}, title = {A comprehensive review of biological and genetic control approaches for leishmaniasis vector sand flies; emphasis towards promoting tools for integrated vector management.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012795}, doi = {10.1371/journal.pntd.0012795}, pmid = {39869587}, issn = {1935-2735}, mesh = {Animals ; *Psychodidae/genetics/parasitology ; *Leishmaniasis/prevention & control/transmission ; *Insect Vectors/genetics/parasitology ; Insect Control/methods ; Humans ; Gene Editing/methods ; Leishmania/genetics ; CRISPR-Cas Systems ; Pest Control, Biological/methods ; }, abstract = {BACKGROUND: Leishmaniasis is a health problem in many regions with poor health and poor life resources. According to the World Health Organization (WHO), an estimated 700,000-1 million new cases arise annually. Effective control of sand fly vector populations is crucial for reducing the transmission of this disease. Therefore, this review aims to comprehensively examine and evaluate the current methods for controlling sand fly populations, focusing on biological and gene drive techniques.
METHODS AND FINDINGS: A detailed, comprehensive literature search was carried out using databases including Google Scholar, PubMed, ScienceDirect, and the National Library of Medicine (NIH). These searches were done using specific keywords related to the field of study. This current review identified several promising methods, including genetically modified sand flies, using transgenic approaches by taking advanced gene editing tools like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) and genetic modification of symbiotic microorganisms for controlling sand fly populations, which appeared to be proven under laboratory and field settings.
CONCLUSION: Genetic control approaches have many benefits over chemical control, including long-lasting effects on targets, high specificity, and less environmental impact. Advances in genetic engineering technologies, particularly CRISPR/Cas9, sterile insect techniques, and gene drive insect modification, offer new avenues for precise and efficient sand fly management. Future research should prioritize optimizing rearing and sterilization techniques, conducting controlled field trials, and fostering collaboration across disciplines to realize the potential of genetic control strategies in combating leishmaniasis.}, }
@article {pmid39868861, year = {2025}, author = {Shahrokhtash, A and Sivertsen, MVT and Laursen, SH and Sutherland, DS}, title = {Nanoscale Cellular Traction Force Quantification: CRISPR-Cas12a Supercharged DNA Tension Sensors in Nanoclustered Ligand Patterns.}, journal = {ACS applied materials & interfaces}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsami.4c18358}, pmid = {39868861}, issn = {1944-8252}, abstract = {High-throughput measurement of cellular traction forces at the nanoscale remains a significant challenge in mechanobiology, limiting our understanding of how cells interact with their microenvironment. Here, we present a novel technique for fabricating protein nanopatterns in standard multiwell microplate formats (96/384-wells), enabling the high-throughput quantification of cellular forces using DNA tension gauge tethers (TGTs) amplified by CRISPR-Cas12a. Our method employs sparse colloidal lithography to create nanopatterned surfaces with feature sizes ranging from sub 100 to 800 nm on transparent, planar, and fully PEGylated substrates. These surfaces allow for the orthogonal immobilization of two different proteins or biomolecules using click-chemistry, providing precise spatial control over cellular signaling cues. We demonstrate the robustness and versatility of this platform through imaging techniques, including total internal reflection fluorescence microscopy, confocal laser scanning microscopy, and high-throughput imaging. Applying this technology, we measured the early stage mechanical forces exerted by 3T3 fibroblasts across different nanoscale features, detecting forces ranging from 12 to 56 pN. By integrating the Mechano-Cas12a Assisted Tension Sensor (MCATS) system, we achieved rapid and high-throughput quantification of cellular traction forces, analyzing over 2 million cells within minutes. Our findings reveal that nanoscale clustering of integrin ligands significantly influences the mechanical responses of cells. This platform offers a powerful tool for mechanobiology research, facilitating the study of cellular forces and mechanotransduction pathways in a high-throughput manner compatible with standard cell culture systems.}, }
@article {pmid39868543, year = {2025}, author = {}, title = {Correction to 'DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems'.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkaf024}, pmid = {39868543}, issn = {1362-4962}, }
@article {pmid39868533, year = {2025}, author = {Hu, Z and Ling, S and Duan, J and Yu, Z and Che, Y and Wang, S and Zhang, S and Zhang, X and Li, Z}, title = {Proximity-activated guide RNA of CRISPR-Cas12a for programmable diagnostic detection and gene regulation.}, journal = {Nucleic acids research}, volume = {53}, number = {3}, pages = {}, doi = {10.1093/nar/gkaf017}, pmid = {39868533}, issn = {1362-4962}, support = {2022YFF0710200//National Science and Technology Major Project of the Ministry of Science and Technology of China/ ; 22304007//National Natural Science Foundation of China/ ; 2252014//Beijing Municipal Natural Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *Escherichia coli/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Gene Editing/methods ; Aptamers, Nucleotide/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Gene Expression Regulation ; Theophylline ; HEK293 Cells ; Gene Knockout Techniques ; Bacterial Proteins ; }, abstract = {The flexibility and programmability of CRISPR-Cas technology have made it one of the most popular tools for biomarker diagnostics and gene regulation. Especially, the CRISPR-Cas12 system has shown exceptional clinical diagnosis and gene editing capabilities. Here, we discovered that although the top loop of the 5' handle of guide RNA can undergo central splitting, deactivating CRISPR-Cas12a, the segments can dramatically restore CRISPR function through nucleic acid self-assembly or interactions with small molecules and aptamers. This discovery forms the basis of an engineered Cas12a system with a programmable proximity-activated guide RNA (PARC-Cas12a) that links targets of interest to dsDNA. Leveraging the efficient trans- and cis-cleavage of Cas12, our findings further inspired a detection platform design for RNAs or non-nucleic acid biomarkers, enabling highly sensitive and multiplexed analysis. We further demonstrated the feasibility of RNA-controllable gene knockout/knockdown in Escherichia coli. Notably, we successfully validated the gene regulatory capabilities of the PARC-Cas12a system within mammalian cell systems by utilizing the classical theophylline molecule-aptamer system. Our results introduce a programmable toolbox for precise diagnostics and cell regulation, allowing the development of versatile diagnostic tools, complex synthetic biological circuits, and cellular biosensors.}, }
@article {pmid39866497, year = {2025}, author = {Owaid, HA and Al-Ouqaili, MTS}, title = {Molecular characterization and genome sequencing of selected highly resistant clinical isolates of Pseudomonas aeruginosa and its association with the clustered regularly interspaced palindromic repeat/Cas system.}, journal = {Heliyon}, volume = {11}, number = {1}, pages = {e41670}, pmid = {39866497}, issn = {2405-8440}, abstract = {The presence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in the superbug Pseudomonas (P) aeruginosa presents a unique opportunity to precisely target and edit bacterial genomes to modify their drug resistance. The objective was to detect the prevalence of CRISPR in extensively and pan-drug-resistant Pseudomonas aeruginosa and to determine the utility of whole-genome sequencing (WGS) for the analysis of the entire genome for such strains. The antimicrobial susceptibilities of one hundred isolates were assessed using the antibiotic susceptibility test (AST) card of the VITEK system. The presence of the CRISPR/Cas system was determined via specific primers using conventional polymerase chain reaction (PCR). Further, WGS was conducted using a DNA nanoball sequencing platform via BGI-Tech for the isolates of interest. Out of 54 resistant Pseudomonas aeruginosa isolates, 33 (33.0 %) were metallo-β-lactamase producers. Cas1, Cas3, CRISPR1, and CRISPR2 were positive in 6.0 % of isolates, while incomplete CRISPR1-Cas systems alone were found in 15.0 %. Also, CRISPR2-type was found intact in 26 % of isolates. The prevalence of resistance to antimicrobials in P. aeruginosa isolates was significantly greater in the CRISPR/Cas-negative group compared to the CRISPR/Cas-positive. Significant relationships for variables were examined using Fisher's exact tests using Chi-squared and a P-value of <0.05 as a statistical threshold. Further, on examination of CRs as a collective entity, encompassing both extensive drug resistance (XDR) and pan-drug resistance (PDR), it becomes evident that the vast majority of these strains (n = 29; 87.8 %) lacked CRISPR/Cas systems. In phylogenic analysis, PDR-P. aeruginosa revealed a very close evolutionary relationship with those originating from Kazakhstan, while XDR was globally unique. Further, the entire genome showed the presence of unique virulence and resistant pseudomonal genes. The CRISPR/Cas system and drug resistance are antagonistic to one another. XDR and PDR P. aeruginosa represent a potential threat to public health and contribute to the seriousness of associated illnesses by leading to resistant infections. Further, WGS for the two strains revealed resistance to multiple antibiotics. It is important to examine specific antimicrobial resistance (AMR) pathways, which suggests that a significant number of resistant genes in these isolates indicate a loss of CRISPR genes in the two strains. Furthermore, the WGS approach can lead to a better understanding of the genomic mechanism of pseudomonal resistance to antibiotics.}, }
@article {pmid39866228, year = {2025}, author = {Lin, HW and Lee, PY and Chang, YS and Chang, MS}, title = {Loss of Arhgap39 facilitates cell migration and invasion in murine hepatocellular cancer cells.}, journal = {Oncology research}, volume = {33}, number = {2}, pages = {493-503}, pmid = {39866228}, issn = {1555-3906}, mesh = {Animals ; *Cell Movement ; Mice ; *GTPase-Activating Proteins/metabolism/genetics ; *Carcinoma, Hepatocellular/pathology/metabolism/genetics ; *Liver Neoplasms/pathology/metabolism/genetics ; *Neoplasm Invasiveness ; Cell Line, Tumor ; Humans ; Mice, Knockout ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Rho GTPases are essential regulators for cellular movement and intracellular membrane trafficking. Their enzymatic activities fluctuate between active GTP-bound and inactive GDP-bound states regulated by GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Arhgap39/Vilse/Porf-2 is a newly identified GAP. The role of Arhgap39 in migration and invasion has not been addressed thoroughly.
METHODS: The Arhgap39 gene was knocked out by Crispr-Cas9 gene editing in mouse Hepa1-6 and Hepa-1c1c7 cells to analyze the impact of Arhgap39 depletion on migration and invasion.
RESULTS: Loss of Arhgap39 noticeably increased the migration and invasive potential. Purified Arhgap39 recombinant protein facilitated the hydrolysis of GTP in RhoA and Rac1 in vitro. RNA-seq analysis revealed that matrix metalloproteinase 13 (MMP13) and Laminin subunit beta 1 (LAMB1) were increased in Arhgap39[-/-] cells. We further crossed Arhgap39fl/fl with KrasLSL-G12D and p53fl/fl mice under the control of albumin-Cre recombinase to induce the spontaneous development of hepatocellular carcinomas. Intriguingly, the expression levels of MMP13 and the overall survival in Alb-Cre_KrasLSL-G12D_p53fl/fl_Arhgap39fl/fl (KPA) mice were comparable to control Alb-Cre_KrasLSL-G12D_p53fl/fl (KP) mice. The cell migration and invasion of KPA mice were also similar to those of control KP mice.
CONCLUSION: Arhgap39 loss could modulate the migration and invasion in some hepatocellular cancer cells, but not in those isolated from KPA mice.}, }
@article {pmid39866131, year = {2025}, author = {Redel, BK and Lee, K}, title = {Response to Cook et al. re: Novel Off-Targeting Events Identified After Genome Wide Analysis of CRISPR-Cas Edited Pig.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2025.0003}, pmid = {39866131}, issn = {2573-1602}, }
@article {pmid39862084, year = {2025}, author = {Szabó, Á and Galla, Z and Spekker, E and Szűcs, M and Martos, D and Takeda, K and Ozaki, K and Inoue, H and Yamamoto, S and Toldi, J and Ono, E and Vécsei, L and Tanaka, M}, title = {Oxidative and Excitatory Neurotoxic Stresses in CRISPR/Cas9-Induced Kynurenine Aminotransferase Knockout Mice: A Novel Model for Despair-Based Depression and Post-Traumatic Stress Disorder.}, journal = {Frontiers in bioscience (Landmark edition)}, volume = {30}, number = {1}, pages = {25706}, doi = {10.31083/FBL25706}, pmid = {39862084}, issn = {2768-6698}, support = {K138125//National Research, Development, and Innovation Office-NKFIH/ ; 2022/5S729//SZTE SZAOK-KKA/ ; //HUN-REN Hungarian Research Network/ ; JPJSBP120203803//JSPS Joint Research Projects under Bilateral Programs/ ; }, mesh = {Animals ; *Stress Disorders, Post-Traumatic/genetics/metabolism ; *Transaminases/genetics/metabolism ; *Mice, Knockout ; Male ; *Oxidative Stress ; *Disease Models, Animal ; *CRISPR-Cas Systems ; *Depression/genetics/metabolism/etiology ; Mice ; Kynurenine/metabolism ; Kynurenic Acid/metabolism ; Tryptophan/metabolism ; Serotonin/metabolism ; Mice, Inbred C57BL ; }, abstract = {BACKGROUNDS: Memory and emotion are especially vulnerable to psychiatric disorders such as post-traumatic stress disorder (PTSD), which is linked to disruptions in serotonin (5-HT) metabolism. Over 90% of the 5-HT precursor tryptophan (Trp) is metabolized via the Trp-kynurenine (KYN) metabolic pathway, which generates a variety of bioactive molecules. Dysregulation of KYN metabolism, particularly low levels of kynurenic acid (KYNA), appears to be linked to neuropsychiatric disorders. The majority of KYNA is produced by the aadat (kat2) gene-encoded mitochondrial kynurenine aminotransferase (KAT) isotype 2. Little is known about the consequences of deleting the KYN enzyme gene.
METHODS: In CRISPR/Cas9-induced aadat knockout (kat2[-/-]) mice, we examined the effects on emotion, memory, motor function, Trp and its metabolite levels, enzyme activities in the plasma and urine of 8-week-old males compared to wild-type mice.
RESULTS: Transgenic mice showed more depressive-like behaviors in the forced swim test, but not in the tail suspension, anxiety, or memory tests. They also had fewer center field and corner entries, shorter walking distances, and fewer jumping counts in the open field test. Plasma metabolite levels are generally consistent with those of urine: antioxidant KYNs, 5-hydroxyindoleacetic acid, and indole-3-acetic acid levels were lower; enzyme activities in KATs, kynureninase, and monoamine oxidase/aldehyde dehydrogenase were lower, but kynurenine 3-monooxygenase was higher; and oxidative stress and excitotoxicity indices were higher. Transgenic mice displayed depression-like behavior in a learned helplessness model, emotional indifference, and motor deficits, coupled with a decrease in KYNA, a shift of Trp metabolism toward the KYN-3-hydroxykynurenine pathway, and a partial decrease in the gut microbial Trp-indole pathway metabolite.
CONCLUSIONS: This is the first evidence that deleting the aadat gene induces depression-like behaviors uniquely linked to experiences of despair, which appear to be associated with excitatory neurotoxic and oxidative stresses. This may lead to the development of a double-hit preclinical model in despair-based depression, a better understanding of these complex conditions, and more effective therapeutic strategies by elucidating the relationship between Trp metabolism and PTSD pathogenesis.}, }
@article {pmid39861899, year = {2025}, author = {Sugiokto, FG and Li, R}, title = {Targeting EBV Episome for Anti-Cancer Therapy: Emerging Strategies and Challenges.}, journal = {Viruses}, volume = {17}, number = {1}, pages = {}, pmid = {39861899}, issn = {1999-4915}, support = {R01 AI141410/AI/NIAID NIH HHS/United States ; R01AI141410//National Institute of Allergy and Infectious Diseases/ ; }, mesh = {Humans ; *Herpesvirus 4, Human/genetics ; *Neoplasms/therapy ; *Gene Editing/methods ; *Epstein-Barr Virus Infections/virology/therapy ; Plasmids/genetics ; Genome, Viral ; Animals ; CRISPR-Cas Systems ; }, abstract = {As a ubiquitous human pathogen, the Epstein-Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.}, }
@article {pmid39710780, year = {2025}, author = {Cui, S and Wang, K and Yang, Y and Lv, X and Li, X}, title = {An integrated and paper-based microfluidic system employing LAMP-CRISPR and equipped with a portable device for simultaneous detection of pathogens.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {4}, pages = {785-797}, pmid = {39710780}, issn = {1618-2650}, support = {JCKY2021602B009//Defense Industrial Technology Development Program/ ; }, mesh = {*Paper ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; CRISPR-Cas Systems ; Humans ; Molecular Diagnostic Techniques/instrumentation/methods ; Lab-On-A-Chip Devices ; Microfluidic Analytical Techniques/instrumentation/methods ; Bacteria/isolation & purification/genetics ; Equipment Design ; Limit of Detection ; }, abstract = {Point-of-care testing methods are essential for the large-scale diagnosis and monitoring of bacterial infections. This study introduces an integrated platform designed for the simultaneous detection of pathogenic bacteria. Users can simply inject samples into the system, which then conducts the entire procedure in a fully automated manner, eliminating the need for external power sources, all within 60 min or less. The innovative paper-based microfluidic system is capable of lysing bacteria and integrating loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12a system, achieving this with minimal reagent usage on a single piece of paper. The reaction reagents are pre-fabricated as freeze-dried powder on the paper, allowing for long-term storage. A portable and cost-effective detection device has been designed to provide stable temperature control and analyze fluorescent signals, complementing the paper-based microfluidic system. This compact device measures 150 × 150 × 100 mm, weighs less than 1.8 kg, has an average power consumption of under 15 W, and supports external power supply. The device utilizes non-contact QR codes for information transmission, ensuring functionality even in areas without Internet connectivity. This platform is capable of simultaneously detecting five typical pathogenic microorganisms, with a detection limit of 1 copy/μL. It boasts several advantages, including miniaturization, lightweight design, low power consumption, portability, affordability, rapid detection, and ease of operation, making it highly suitable for on-site detection.}, }
@article {pmid39701542, year = {2025}, author = {Oppman, AM and Paradee, WJ and Narayanan, NS and Kim, YC}, title = {Generation and validation of a D1 dopamine receptor Flpo knock-in mouse.}, journal = {Journal of neuroscience methods}, volume = {415}, number = {}, pages = {110345}, doi = {10.1016/j.jneumeth.2024.110345}, pmid = {39701542}, issn = {1872-678X}, support = {R01 NS120987/NS/NINDS NIH HHS/United States ; }, mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice ; *Mice, Transgenic ; Gene Knock-In Techniques/methods ; Brain/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Male ; Neurons/metabolism ; }, abstract = {BACKGROUND: Dopamine is a powerful neuromodulator of diverse brain functions, including movement, motivation, reward, and cognition. D1-type dopamine receptors (D1DRs) are the most prevalently expressed dopamine receptors in the brain. Neurons expressing D1DRs are heterogeneous and involve several subpopulations. Although these neurons can be studied with BAC-transgenic rodents, these models have some limitations especially when considering their integration with conditional or intersectional genetic tools.
NEW METHOD: We developed a novel Drd1-P2A-Flpo (Drd1-Flpo) mouse line in which the Flpo gene was knocked in immediately after the Drd1 gene using CRISPR-Cas9. We validated the Drd1-Flpo line by confirming Flp expression and functionality specific to D1DR+ neurons with immunohistochemistry and in situ hybridization.
The Drd1-Flpo line is a useful resource for studying subpopulations of D1DR+ neurons with intersectional genetic tools.
CONCLUSIONS: We demonstrated brain-wide GFP expression driven by Drd1-Flpo, suggesting that this mouse line may be useful for comprehensive anatomical and functional studies in many brain regions. The Drd1-Flpo model will advance the study of dopaminergic signaling by providing a new tool for investigating the diverse roles of D1DR+ neurons and their subpopulations in brain disease.}, }
@article {pmid39661735, year = {2025}, author = {Baysal, C and Kausch, AP and Cody, JP and Altpeter, F and Voytas, DF}, title = {Rapid and efficient in planta genome editing in sorghum using foxtail mosaic virus-mediated sgRNA delivery.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {2}, pages = {e17196}, doi = {10.1111/tpj.17196}, pmid = {39661735}, issn = {1365-313X}, support = {DE-SC0018420//Biological and Environmental Research/ ; }, mesh = {*Sorghum/genetics/virology ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Lyases/genetics/metabolism ; Oxidoreductases/genetics/metabolism ; Potexvirus/genetics ; Plant Viruses ; }, abstract = {The requirement of in vitro tissue culture for the delivery of gene editing reagents limits the application of gene editing to commercially relevant varieties of many crop species. To overcome this bottleneck, plant RNA viruses have been deployed as versatile tools for in planta delivery of recombinant RNA. Viral delivery of single-guide RNAs (sgRNAs) to transgenic plants that stably express CRISPR-associated (Cas) endonuclease has been successfully used for targeted mutagenesis in several dicotyledonous and few monocotyledonous plants. Progress with this approach in monocotyledonous plants is limited so far by the availability of effective viral vectors. We engineered a set of foxtail mosaic virus (FoMV) and barley stripe mosaic virus (BSMV) vectors to deliver the fluorescent protein AmCyan to track viral infection and movement in Sorghum bicolor. We further used these viruses to deliver and express sgRNAs to Cas9 and Green Fluorescent Protein (GFP) expressing transgenic sorghum lines, targeting Phytoene desaturase (PDS), Magnesium-chelatase subunit I (MgCh), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, orthologs of maize Lemon white1 (Lw1) or GFP. The recombinant BSMV did neither infect sorghum nor deliver or express AmCyan and sgRNAs. In contrast, the recombinant FoMV systemically spread throughout sorghum plants and induced somatic mutations with frequencies reaching up to 60%. This mutagenesis led to visible phenotypic changes, demonstrating the potential of FoMV for in planta gene editing and functional genomics studies in sorghum.}, }
@article {pmid39861013, year = {2025}, author = {Yibar, A and Duman, M and Ay, H and Ajmi, N and Tasci, G and Gurler, F and Guler, S and Morick, D and Saticioglu, IB}, title = {Genomic Insight into Vibrio Isolates from Fresh Raw Mussels and Ready-to-Eat Stuffed Mussels.}, journal = {Pathogens (Basel, Switzerland)}, volume = {14}, number = {1}, pages = {}, doi = {10.3390/pathogens14010052}, pmid = {39861013}, issn = {2076-0817}, support = {TOA-2022-668 and TGA-2024-1841//Bursa Uludağ Üni̇versi̇tesi̇/ ; }, mesh = {Animals ; *Vibrio/genetics/isolation & purification/pathogenicity ; *Bivalvia/microbiology ; *Genome, Bacterial/genetics ; *Virulence Factors/genetics ; Genomics ; Food Microbiology ; Shellfish/microbiology ; Phylogeny ; }, abstract = {Consuming raw or undercooked mussels can lead to gastroenteritis and septicemia due to Vibrio contamination. This study analyzed the prevalence, density, species diversity, and molecular traits of Vibrio spp. in 48 fresh raw wild mussels (FRMs) and 48 ready-to-eat stuffed mussels (RTE-SMs) through genome analysis, assessing health risks. The results showed Vibrio prevalence rates of 12.5% in FRMs and 4.2% in RTE-SMs, with V. alginolyticus as the most common species (46.7%). It was determined that the seasonal distribution of Vibrio spp. prevalence in the samples was higher in the summer months. The genome sizes of the Vibrio spp. ranged from approximately 3.9 to 6.1 Mb, with the GC contents varying between 41.9% and 50.4%. A total of 22 virulence factor (VF) classes and up to six antimicrobial resistance (AMR) genes were detected in different Vibrio species. The presence of nine different biosynthetic gene clusters (BGCs), 27 prophage regions, and eight CRISPR/Cas systems in 15 Vibrio strains provides information about their potential pathogenicity, survival strategies, and adaptation to different habitats. Overall, this study provides a comprehensive understanding of the genomic diversity of Vibrio spp. isolated from FRM and RTE-SM samples, shedding light on the prevalence, pathogenicity, and toxicity mechanisms of Vibrio-induced gastroenteritis.}, }
@article {pmid39858940, year = {2025}, author = {Ma, J and Lu, Z}, title = {Developing a Versatile Arsenal: Novel Antimicrobials as Offensive Tools Against Pathogenic Bacteria.}, journal = {Microorganisms}, volume = {13}, number = {1}, pages = {}, doi = {10.3390/microorganisms13010172}, pmid = {39858940}, issn = {2076-2607}, support = {31970101//the National Natural Science Foundation of China/ ; }, abstract = {The pervasive and often indiscriminate use of antibiotics has accelerated the emergence of drug-resistant bacterial strains, thus presenting an acute threat to global public health. Despite a growing acknowledgment of the severity of this crisis, the current suite of strategies to mitigate antimicrobial resistance remains markedly inadequate. This paper asserts the paramount need for the swift development of groundbreaking antimicrobial strategies and provides a comprehensive review of an array of innovative techniques currently under scrutiny. Among these, nano-antimicrobials, antimicrobials derived from ribosomal proteins, CRISPR/Cas-based systems, agents that undermine bacterial bioenergetics, and antimicrobial polysaccharides hold particular promise. This analysis gives special attention to CRISPR/Cas-based antimicrobials, scrutinizing their underlying mechanisms, exploring their potential applications, delineating their distinct advantages, and noting their likely limitations. Furthermore, we extend our exploration by proposing theoretical advancements in antimicrobial technology and evaluating feasible methods for the effective delivery of these agents. This includes leveraging these advances for broader biomedical applications, potentially revolutionizing how we confront bacterial pathogens in the future, and laying a foundation for extended research in multimodal therapeutic strategies.}, }
@article {pmid39858819, year = {2024}, author = {Zhang, Z and Wei, M and Jia, B and Yuan, Y}, title = {Recent Advances in Antimicrobial Resistance: Insights from Escherichia coli as a Model Organism.}, journal = {Microorganisms}, volume = {13}, number = {1}, pages = {}, doi = {10.3390/microorganisms13010051}, pmid = {39858819}, issn = {2076-2607}, support = {2018YFA0903700//National Natural Science Foundation of China/ ; }, abstract = {Antimicrobial resistance (AMR) represents a critical global health threat, and a thorough understanding of resistance mechanisms in Escherichia coli is needed to guide effective treatment interventions. This review explores recent advances for investigating AMR in E. coli, including machine learning for resistance pattern analysis, laboratory evolution to generate resistant mutants, mutant library construction, and genome sequencing for in-depth characterization. Key resistance mechanisms are discussed, including drug inactivation, target modification, altered transport, and metabolic adaptation. Additionally, we highlight strategies to mitigate the spread of AMR, such as dynamic resistance monitoring, innovative therapies like phage therapy and CRISPR-Cas technology, and tighter regulation of antibiotic use in animal production systems. This review provides actionable insights into E. coli resistance mechanisms and identifies promising directions for future antibiotic development and AMR management.}, }
@article {pmid39858595, year = {2025}, author = {Dansereau, SJ and Cui, H and Dartawan, RP and Sheng, J}, title = {The Plethora of RNA-Protein Interactions Model a Basis for RNA Therapies.}, journal = {Genes}, volume = {16}, number = {1}, pages = {}, doi = {10.3390/genes16010048}, pmid = {39858595}, issn = {2073-4425}, support = {1R01GM143749-22A1/NH/NIH HHS/United States ; }, mesh = {Humans ; *RNA/metabolism/genetics ; Animals ; CRISPR-Cas Systems ; Aptamers, Nucleotide/genetics/metabolism ; Genetic Therapy/methods ; RNA, Small Interfering/genetics/therapeutic use/metabolism ; RNA-Binding Proteins/metabolism/genetics ; }, abstract = {The notion of RNA-based therapeutics has gained wide attractions in both academic and commercial institutions. RNA is a polymer of nucleic acids that has been proven to be impressively versatile, dating to its hypothesized RNA World origins, evidenced by its enzymatic roles in facilitating DNA replication, mRNA decay, and protein synthesis. This is underscored through the activities of riboswitches, spliceosomes, ribosomes, and telomerases. Given its broad range of interactions within the cell, RNA can be targeted by a therapeutic or modified as a pharmacologic scaffold for diseases such as nucleotide repeat disorders, infectious diseases, and cancer. RNA therapeutic techniques that have been researched include, but are not limited to, CRISPR/Cas gene editing, anti-sense oligonucleotides (ASOs), siRNA, small molecule treatments, and RNA aptamers. The knowledge gleaned from studying RNA-centric mechanisms will inevitably improve the design of RNA-based therapeutics. Building on this understanding, we explore the physiological diversity of RNA functions, examine specific dysfunctions, such as splicing errors and viral interactions, and discuss their therapeutic implications.}, }
@article {pmid39857007, year = {2025}, author = {Lakshmanan, K and Liu, BM}, title = {Impact of Point-of-Care Testing on Diagnosis, Treatment, and Surveillance of Vaccine-Preventable Viral Infections.}, journal = {Diagnostics (Basel, Switzerland)}, volume = {15}, number = {2}, pages = {}, doi = {10.3390/diagnostics15020123}, pmid = {39857007}, issn = {2075-4418}, support = {P30AI117970; U54AI150225/GF/NIH HHS/United States ; }, abstract = {With the advent of a variety of vaccines against viral infections, there are multiple viruses that can be prevented via vaccination. However, breakthrough infections or uncovered strains can still cause vaccine-preventable viral infections (VPVIs). Therefore, timely diagnosis, treatment, and surveillance of these viruses is critical to patient care and public health. Point-of-care (POC) viral diagnostics tools have brought significant improvements in the detection and management of VPVIs. These cutting-edge technologies enable prompt and accurate results, enhancing patient care by facilitating timely treatment decisions. This review delves into the advancements in POC testing, including antigen/antibody detection and molecular assays, while focusing on their impact on the diagnosis, treatment, and surveillance of VPVIs such as mpox, viral hepatitis, influenza, flaviviruses (dengue, Zika, and yellow fever virus), and COVID-19. The role of POC tests in monitoring viral infection is crucial for tracking disease progression and managing outbreaks. Furthermore, the application of POC diagnostics has shown to be vital for public health strategies. In this review, we also highlight emerging POC technologies such as CRISPR-based diagnostics and smartphone-integrated POC devices, which have proven particularly beneficial in resource-limited settings. We underscore the importance of continued research to optimize these diagnostic tools for wider global use for mpox, viral hepatitis, influenza, dengue, and COVID-19, while also addressing current challenges related to their sensitivity, specificity, availability, efficiency, and more.}, }
@article {pmid39856077, year = {2025}, author = {Xu, X and Chen, J and Wang, Y and Liu, Y and Zhang, Y and Yang, J and Yang, X and Chen, B and He, Z and Champer, J}, title = {Gene drive-based population suppression in the malaria vector Anopheles stephensi.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {1007}, pmid = {39856077}, issn = {2041-1723}, support = {32302455//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Anopheles/genetics ; Female ; *Mosquito Vectors/genetics ; *Malaria/transmission/prevention & control ; Male ; *Gene Drive Technology/methods ; Mosquito Control/methods ; Animals, Genetically Modified ; Alleles ; CRISPR-Cas Systems ; }, abstract = {Gene drives are alleles that can bias the inheritance of specific traits in target populations for the purpose of modification or suppression. Here, we construct a homing suppression drive in the major urban malaria vector Anopheles stephensi targeting the female-specific exon of doublesex, incorporating two gRNAs and a nanos-Cas9 to reduce functional resistance and improve female heterozygote fitness. Our results show that the drive was recessive sterile in both females and males, with various intersex phenotypes in drive homozygotes. Both male and female drive heterozygotes show only moderate drive conversion, indicating that the nanos promoter has lower activity in A. stephensi than in Anopheles gambiae. By amplicon sequencing, we detect a very low level of resistance allele formation. Combination of the homing suppression drive and a vasa-Cas9 line boosts the drive conversion rate of the homing drive to 100%, suggesting the use of similar systems for population suppression in a continuous release strategy with a lower release rate than SIT or fsRIDL techniques. This study contributes valuable insights to the development of more efficient and environmentally friendly pest control tools aimed at disrupting disease transmission.}, }
@article {pmid39856062, year = {2025}, author = {Pena, IA and Shi, JS and Chang, SM and Yang, J and Block, S and Adelmann, CH and Keys, HR and Ge, P and Bathla, S and Witham, IH and Sienski, G and Nairn, AC and Sabatini, DM and Lewis, CA and Kory, N and Vander Heiden, MG and Heiman, M}, title = {SLC25A38 is required for mitochondrial pyridoxal 5'-phosphate (PLP) accumulation.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {978}, pmid = {39856062}, issn = {2041-1723}, mesh = {*Mitochondria/metabolism ; *Pyridoxal Phosphate/metabolism ; Humans ; *Glycine Hydroxymethyltransferase/metabolism/genetics ; Cell Proliferation ; CRISPR-Cas Systems ; Mitochondrial Proteins/metabolism/genetics ; Anemia, Sideroblastic/metabolism/genetics ; Vitamin B 6/metabolism ; Cell Line, Tumor ; Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Genetic Diseases, X-Linked ; }, abstract = {Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.}, }
@article {pmid39856035, year = {2025}, author = {Padilla, MS and Mrksich, K and Wang, Y and Haley, RM and Li, JJ and Han, EL and El-Mayta, R and Kim, EH and Dias, S and Gong, N and Teerdhala, SV and Han, X and Chowdhary, V and Xue, L and Siddiqui, Z and Yamagata, HM and Kim, D and Yoon, IC and Wilson, JM and Radhakrishnan, R and Mitchell, MJ}, title = {Branched endosomal disruptor (BEND) lipids mediate delivery of mRNA and CRISPR-Cas9 ribonucleoprotein complex for hepatic gene editing and T cell engineering.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {996}, pmid = {39856035}, issn = {2041-1723}, support = {T90DE030854//U.S. Department of Health & Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; }, mesh = {*Gene Editing/methods ; *Endosomes/metabolism ; *Lipids/chemistry ; *RNA, Messenger/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/chemistry ; *T-Lymphocytes/metabolism ; Ribonucleoproteins/metabolism ; Liver/metabolism ; Animals ; Cell Engineering/methods ; Gene Transfer Techniques ; Transfection/methods ; Mice ; Liposomes ; }, abstract = {Lipid nanoparticles (LNPs) are the preeminent non-viral drug delivery vehicle for mRNA-based therapies. Immense effort has been placed on optimizing the ionizable lipid (IL) structure, which contains an amine core conjugated to lipid tails, as small molecular adjustments can result in substantial changes in the overall efficacy of the resulting LNPs. However, despite some advancements, a major barrier for LNP delivery is endosomal escape. Here, we develop a platform for synthesizing a class of branched ILs that improve endosomal escape. These compounds incorporate terminally branched groups that increase hepatic mRNA and ribonucleoprotein complex delivery and gene editing efficiency as well as T cell transfection compared to non-branched lipids. Through an array of complementary experiments, we determine that our lipid architecture induces greater endosomal penetration and disruption. This work provides a scheme to generate a class of ILs for both mRNA and protein delivery.}, }
@article {pmid39853972, year = {2025}, author = {Fu, R and Hou, J and Wang, Z and Zhu, C and Xianyu, Y}, title = {A CRISPR-Cas and Argonaute-Driven Two-Factor Authentication Strategy for Information Security.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.4c17560}, pmid = {39853972}, issn = {1936-086X}, abstract = {The escalating growth in computing power and the advent of quantum computing present a critical threat to the security of modern cryptography. Two-factor authentication strategies can effectively resist brute-force attacks to improve the security of access control. Herein, we proposed a two-factor and two-authentication entity strategy based on the trans-cleavage activity of CRISPR-Cas and the "dual-step" sequence-specific cleavage of Pyrococcus furiosus Argonaute. In this strategy, the output of authentication entity 1 acted as a component to operate authentication entity 2, thus enabling a role-based molecular model that implemented access control for the three roles. To further enhance information security, we designed knowledge suppression factors to constitute the command library and possession suppression factors to resist brute-force attacks. This study will promote the development of advanced molecular access control and its applications in biomedical diagnostics and data security.}, }
@article {pmid39851566, year = {2025}, author = {Pohl, KA and Zhang, X and Ji, JJ and Stiles, L and Sadun, AA and Yang, XJ}, title = {Derivation and Characterization of Isogenic OPA1 Mutant and Control Human Pluripotent Stem Cell Lines.}, journal = {Cells}, volume = {14}, number = {2}, pages = {}, doi = {10.3390/cells14020137}, pmid = {39851566}, issn = {2073-4409}, support = {2R01EY026319, F31EY033242, P30EY000331/GF/NIH HHS/United States ; }, mesh = {Humans ; *GTP Phosphohydrolases/metabolism/genetics ; *Mutation/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Optic Atrophy, Autosomal Dominant/genetics/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Retinal Ganglion Cells/metabolism ; Pluripotent Stem Cells/metabolism/cytology ; Mitochondria/metabolism ; }, abstract = {Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene, which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion, proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed, human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs, DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs, we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition, CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient's iPSCs. In comparison to the isogenic controls, the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore, OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration.}, }
@article {pmid39716581, year = {2025}, author = {Kalaichelvan, A and Nadarajapillai, K and Sellaththurai, SR and Arachchi, UPE and Kim, MJ and Jung, S and Lee, J}, title = {CRISPR/Cas9-induced knockout of tumor necrosis factor-alpha-type I augments viral infection in zebrafish.}, journal = {Fish & shellfish immunology}, volume = {157}, number = {}, pages = {110092}, doi = {10.1016/j.fsi.2024.110092}, pmid = {39716581}, issn = {1095-9947}, mesh = {Animals ; *Zebrafish/immunology/genetics/virology ; *Novirhabdovirus/physiology ; *CRISPR-Cas Systems ; *Tumor Necrosis Factor-alpha/genetics/immunology/metabolism ; *Gene Knockout Techniques ; Fish Diseases/immunology/virology/genetics ; Hemorrhagic Septicemia, Viral/immunology/genetics/virology ; Immunity, Innate/genetics ; Rhabdoviridae Infections/immunology/veterinary ; Zebrafish Proteins/genetics/immunology ; }, abstract = {Tumor necrosis factor-alpha (TNF-α) is a pleiotropic cytokine with critical roles in inflammation, cell survival, and defense. As a member of the TNF superfamily, it exerts its effects by binding to transmembrane receptors and triggering various downstream signaling pathways. Although TNF-α's involvement in antiviral responses in mammals is well-established, its role in teleost remains poorly understood. This study investigated the contribution of TNF-α1 to antiviral immunity in zebrafish using a tnf-α1[(-/-)] knockout (KO) line. We challenged both wild-type and tnf-α1[(-/-)] zebrafish with viral hemorrhagic septicemia virus (VHSV) at both embryonic and adult stages. Mortality was observed at 4 days post-infection (dpi) in tnf-α1-deficient adult fish challenged with 5 × 10[6] TCID50 (VHSV) and at 5 dpi in adult wild fish challenged with the same concentration. In addition, tnf-α1[(-/-)] KO adult fish reached the maximum mortality of 100 % at 20 dpi, whereas wild adult fish reached 54 % mortality at the same time point. This increased susceptibility to early mortality was associated with a higher viral burden and altered expression of key immune genes, including the pro-inflammatory cytokines il-6 and il-1β, the anti-inflammatory cytokine il-10, and interferon-related genes such as irf1 and ifn-γ. Our findings demonstrate the crucial role of TNF-α1 in antiviral defense mechanisms in zebrafish and provide valuable insights into the functional conservation of TNF-α signaling across vertebrate species. This knowledge may contribute to the development of strategies to combat viral diseases in fish.}, }
@article {pmid39675302, year = {2025}, author = {Sun, Y and Zhang, W and Zhang, H and Zhao, F and Su, L}, title = {CRISPR/Cas13a combined with reverse transcription and RPA for NoV GII.4 monitoring in water environments.}, journal = {Environment international}, volume = {195}, number = {}, pages = {109195}, doi = {10.1016/j.envint.2024.109195}, pmid = {39675302}, issn = {1873-6750}, mesh = {*Norovirus/genetics ; *Environmental Monitoring/methods ; *CRISPR-Cas Systems ; *Reverse Transcription ; RNA, Viral/genetics ; Water Microbiology ; }, abstract = {Water bodies contaminated with the norovirus (NoV) are important vectors for its transmission. Therefore, enhanced monitoring of NoV in aqueous environments plays an active role in preventing diseases. Here, we reverse transcribed viral RNA into cDNA, and then used the constructed RPA-CRISPR/Cas13a-based platform for sensitive and quantitative monitoring of NoV GII.4 in aqueous environments. The use of glycerol as a phase separator and the direct release of nucleic acids from the virus by NaOH significantly enhanced the stability of the assay and reduced its economic cost. This assay is sensitive, specific, and stable. Based on the qualitative detection method, we established a relatively accurate quantitative detection method using the plasmid as a standard. Four water samples, totaling 64 samples, were analyzed using this method and compared with the qPCR method. The results of the two methods showed 100 % concordance with no significant difference in viral load. The entire process of our established method-from viral nucleic acid extraction to the output of the results-was completed in 30 min, much less than the time required for qPCR method. This suggests that the assay can be used as an alternative to qPCR for monitoring the change of NoV GII.4 concentration in water bodies, and shows high potential for application in the immediate detection of viruses in aqueous environments and resource-limited areas.}, }
@article {pmid39581026, year = {2025}, author = {Wang, W and Sun, J and Gao, Y and Jia, XX and Ye, Y and Ren, S and Peng, Y and Han, D and Zhou, H and Gao, Z and Sun, X}, title = {Ultra-sensitive detection of norovirus using a three-in-one CRISPR platform based on a DNA hydrogel and composite functional nanomaterials.}, journal = {Journal of hazardous materials}, volume = {482}, number = {}, pages = {136523}, doi = {10.1016/j.jhazmat.2024.136523}, pmid = {39581026}, issn = {1873-3336}, mesh = {*Norovirus/genetics/isolation & purification ; *Hydrogels/chemistry ; *Biosensing Techniques/methods ; *Colorimetry/methods ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; DNA/chemistry ; Limit of Detection ; Nanostructures/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The ultrasensitive sensor with three optical response mechanisms was proposed for the determination of trace amounts of norovirus using a 3-in-1 GCSNAs (a gap-containing spherical nucleic acid nanoparticles) probe. A simple and highly sensitive three-mode biosensor with Raman, colorimetric, and fluorescence functions was proposed and implemented using the GCSNAs probe and a DNA hydrogel for norovirus detection. When the virus exists, the trans-cleavage activity of CRISPR-Cas12a was activated by double-stranded dsDNA (dsDNA) generated by reverse transcription and recombinase polymerase isothermal amplification (RT-RPA) to degrade the DNA hydrogel/GCSNA composition and release the three-in-one (3-in-1) probe-GCSNA, realising the triple ultrasensitive detection of norovirus. The colorimetric sensing mode allows for semi-quantitative on-site detection, which is visible to the naked eye and the quantitative detection can be achieved by conducting grayscale analysis using the "Colour Grab" function of a smartphone. This new triple sensor achieved the successful quantification of norovirus at concentrations as low as the femtomolar scale with an excellent selectivity and accuracy. Considering the colorimetric properties of rolling circle amplification (RCA)-based DNA hydrogels and GCSNAs, the proposed method has a broad application prospect in virus on-site detection in food. It should be applicable for virus detection in a wide range of fields such, as environmental analysis, medical diagnosis, and food safety. It is anticipated that this mechanism will open new avenues for the development of multimodal analyses and multifunctional sensing platforms for various applications. We anticipate that this sensing mechanism will open up a new way for the development of food safety detection.}, }
@article {pmid39851560, year = {2025}, author = {Bonowicz, K and Jerka, D and Piekarska, K and Olagbaju, J and Stapleton, L and Shobowale, M and Bartosiński, A and Łapot, M and Bai, Y and Gagat, M}, title = {CRISPR-Cas9 in Cardiovascular Medicine: Unlocking New Potential for Treatment.}, journal = {Cells}, volume = {14}, number = {2}, pages = {}, doi = {10.3390/cells14020131}, pmid = {39851560}, issn = {2073-4409}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cardiovascular Diseases/genetics/therapy ; *Genetic Therapy/methods ; Animals ; }, abstract = {Cardiovascular diseases (CVDs) remain a significant global health challenge, with many current treatments addressing symptoms rather than the genetic roots of these conditions. The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering a transformative approach to targeting disease-causing mutations directly. This article examines the potential of CRISPR-Cas9 in the treatment of various CVDs, including atherosclerosis, arrhythmias, cardiomyopathies, hypertension, and Duchenne muscular dystrophy (DMD). The technology's ability to correct single-gene mutations with high precision and efficiency positions it as a groundbreaking tool in cardiovascular therapy. Recent developments have extended the capabilities of CRISPR-Cas9 to include mitochondrial genome editing, a critical advancement for addressing mitochondrial dysfunctions often linked to cardiovascular disorders. Despite its promise, significant challenges remain, including off-target effects, ethical concerns, and limitations in delivery methods, which hinder its translation into clinical practice. This article also explores the ethical and regulatory considerations surrounding gene editing technologies, emphasizing the implications of somatic versus germline modifications. Future research efforts should aim to enhance the accuracy of CRISPR-Cas9, improve delivery systems for targeted tissues, and ensure the safety and efficacy of treatments in the long term. Overcoming these obstacles could enable CRISPR-Cas9 to not only treat but also potentially cure genetically driven cardiovascular diseases, heralding a new era in precision medicine for cardiovascular health.}, }
@article {pmid39849243, year = {2025}, author = {Nawab, S and Ullah, MW and Shah, SB and Zhang, YF and Keerio, HA and Yong, YC}, title = {Recent advances in engineering non-native microorganisms for poly(3-hydroxybutyrate) production.}, journal = {World journal of microbiology & biotechnology}, volume = {41}, number = {2}, pages = {48}, pmid = {39849243}, issn = {1573-0972}, support = {2023M741421//China Postdoctoral Science Foundation/ ; 2022ZB650//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023ZB399//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; BK20220003//Natural Science Foundation of Jiangsu Province/ ; }, mesh = {*Metabolic Engineering/methods ; *Hydroxybutyrates/metabolism ; *Polyesters/metabolism ; Escherichia coli/genetics/metabolism ; Biosynthetic Pathways/genetics ; Synthetic Biology/methods ; CRISPR-Cas Systems ; Bacteria/metabolism/genetics ; Metabolic Networks and Pathways/genetics ; Industrial Microbiology ; Polyhydroxybutyrates ; }, abstract = {Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer that belongs to a group of polymers called polyhydroxyalkanoates (PHAs). PHB can be synthesized from renewable resources, making it a promising alternative to petroleum-derived plastics. It is also considered non-toxic, biodegradable, and biocompatible, which makes it suitable for various applications in the medicine and biomedicine. Many microorganisms biosynthesize and accumulate PHB naturally. However, recent advancements in metabolic engineering and synthetic biology have allowed scientists to engineer non-native microorganisms to produce PHB. This review comprehensively summarizes all non-native microbial hosts used for PHB biosynthesis and discusses different metabolic engineering approaches used to enhance PHB production. These strategies include optimizing the biosynthesis pathway through cofactor engineering, metabolic pathway reconstruction, and cell morphology engineering. Moreover, the CRISPR/Cas9 approach is also used for manipulating the genome of non-host microorganisms to enable them produce PHB. Among non-native microbial hosts, Escherichia coli has been successfully used for industrial-scale PHB production. However, further genetic engineering approaches are needed to make non-native microbial hosts more suitable for large-scale PHB production.}, }
@article {pmid39849074, year = {2025}, author = {Das, S and Unhale, T and Marinach, C and Valeriano Alegria, BDC and Roux, C and Madry, H and Mohand Oumoussa, B and Amino, R and Iwanaga, S and Briquet, S and Silvie, O}, title = {Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2949}, pmid = {39849074}, issn = {2045-2322}, support = {ANR-11-LABX-0024//Agence Nationale de la Recherche/ ; EQU201903007823//Fondation pour la recherche médicale, France/ ; }, mesh = {*Plasmodium berghei/genetics ; *Integrases/metabolism/genetics ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Sirolimus/pharmacology ; Mice ; Malaria/parasitology ; }, abstract = {Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. Another potent tool is the CRISPR/Cas9 technology, which has enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR/Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirmed the role of CLAMP during invasion of erythrocytes.}, }
@article {pmid39804945, year = {2025}, author = {Huang, Y and Pei, S and Lv, X and Yang, F and Gong, X and Li, N and Guo, Y and Feng, Y and Xiao, L}, title = {Stage-specific expression and divergent functions of two insulinase-like proteases associated with host infectivity in Cryptosporidium.}, journal = {PLoS neglected tropical diseases}, volume = {19}, number = {1}, pages = {e0012777}, doi = {10.1371/journal.pntd.0012777}, pmid = {39804945}, issn = {1935-2735}, mesh = {Animals ; *Cryptosporidiosis/parasitology ; Mice ; *Cryptosporidium parvum/genetics/enzymology/pathogenicity ; Mice, Knockout ; Insulysin/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; }, abstract = {BACKGROUND: The determinants of differences in host infectivity among Cryptosporidium species and subtypes are poorly understood. Results from recent comparative genomic studies suggest that gains and losses of multicopy subtelomeric genes encoding insulinase-like proteases (INS-19 and INS-20 in Cryptosporidium parvum and their orthologs in closely related species) may potentially contribute to these differences.
In this study, we investigated the expression and biological function of the INS-19 and INS-20 of C. parvum. CRISPR/Cas9 was used to endogenously tag both genes with the hemagglutinin epitope. Immunofluorescence analysis revealed that INS-19 and INS-20 are expressed at different developmental stages of the pathogen. Although knockout of either had no detectable effect on the in vitro growth of C. parvum, knockout of INS-20, deletion of its multiple domains, or mutation of the active motif in the functional domain reduced the intensity of C. parvum infection in IFN-γ knockout mice. Consistent with this, mice infected with the INS-20-deleted mutant had reduced intestinal damage and parasite burden.
CONCLUSIONS/SIGNIFICANCE: These results suggest that INS-19 and INS-20 have stage-specific expression with distinct biological functions, and that the presence of the INS-20 in zoonotic C. parvum contributes to its infectivity and fitness in mice.}, }
@article {pmid39804925, year = {2025}, author = {Ricou, A and Simon, M and Duflos, R and Azzopardi, M and Roux, F and Budar, F and Camilleri, C}, title = {Identification of novel genes responsible for a pollen killer present in local natural populations of Arabidopsis thaliana.}, journal = {PLoS genetics}, volume = {21}, number = {1}, pages = {e1011451}, doi = {10.1371/journal.pgen.1011451}, pmid = {39804925}, issn = {1553-7404}, mesh = {*Arabidopsis/genetics ; *Pollen/genetics ; *Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Alleles ; DNA Copy Number Variations/genetics ; Mitochondria/genetics/metabolism ; Genes, Plant ; }, abstract = {Gamete killers are genetic loci that distort segregation in the progeny of hybrids because the killer allele promotes the elimination of the gametes that carry the sensitive allele. They are widely distributed in eukaryotes and are important for understanding genome evolution and speciation. We had previously identified a pollen killer in hybrids between two distant natural accessions of Arabidopsis thaliana. This pollen killer involves three genetically linked genes, and we previously reported the identification of the gene encoding the antidote that protects pollen grains from the killer activity. In this study, we identified the two other genes of the pollen killer by using CRISPR-Cas9 induced mutants. These two genes are necessary for the killer activity that we demonstrated to be specific to pollen. The cellular localization of the pollen killer encoded proteins suggests that the pollen killer activity involves the mitochondria. Sequence analyses reveal predicted domains from the same families in the killer proteins. In addition, the C-terminal half of one of the killer proteins is identical to the antidote, and one amino acid, crucial for the antidote activity, is also essential for the killer function. Investigating more than 700 worldwide accessions of A. thaliana, we confirmed that the locus is subject to important structural rearrangements and copy number variation. By exploiting available de novo genomic sequences, we propose a scenario for the emergence of this pollen killer in A. thaliana. Furthermore, we report the co-occurrence and behavior of killer and sensitive genotypes in several local populations, a prerequisite for studying gamete killer evolution in the wild. This highlights the potential of the Arabidopsis model not only for functional studies of gamete killers but also for investigating their evolutionary trajectories at complementary geographical scales.}, }
@article {pmid39661534, year = {2025}, author = {Dhinoja, S and Mary, J and Qaryoute, AA and De Maria, A and Jagadeeswaran, P}, title = {Generation and characterization of zebrafish f9l mutant confirmed that f9l is f10 like gene.}, journal = {Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis}, volume = {36}, number = {1}, pages = {26-33}, doi = {10.1097/MBC.0000000000001337}, pmid = {39661534}, issn = {1473-5733}, mesh = {Animals ; *Zebrafish/genetics ; Zebrafish Proteins/genetics ; Mutation ; CRISPR-Cas Systems ; }, abstract = {AIM: This study aimed to create an f9l mutant zebrafish using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and characterize its coagulation properties to investigate its functional similarity to human FX and explore the potential synergy between f9l and f10 .
METHODS: Three gRNAs targeting exon 8 encoded by the catalytic domain of the f9l gene were injected into 300 single-cell zebrafish embryos using CRISPR/Cas9 technology. DNA from the resulting adults was extracted from tail tips, and PCR was used to detect indels. The identified founder mutant was bred to homozygosity, and functional assays, kinetic Russel viper venom time, bleeding assay in adults, and venous laser injury on larvae were conducted to assess its hemostatic function. Additionally, f10 was knocked down in f9l homozygous embryos using f10 antisense morpholinos to study their interaction by monitoring its survival.
RESULTS: DNA from 60 adults was screened for indels, resulting in a fish with a heritable complex mutation involving one insertion and two deletions in exon 8. The f9l homozygous mutants exhibited impaired F10 activity, mild bleeding after mechanical injury, and developmental deformities in early larval stages. The caudal vein thrombosis assay showed variable occlusion times, indicating a bleeding phenotype with incomplete penetrance. Knocking down f10 in f9l homozygous embryos resulted in 50% mortality within five dpf, compared to f9l homozygous embryos injected with control morpholinos.
CONCLUSION: In summary, we generated f9l knockout and showed it is a paralog to f10. We also found a synergy between f9l and f10 genes, highlighting its importance in hemostasis.}, }
@article {pmid39847570, year = {2025}, author = {Al Sium, SM and Goswami, B and Chowdhury, SF and Naser, SR and Sarkar, MK and Faruq, MJ and Habib, MA and Akter, S and Banu, TA and Sarkar, MMH and Khan, MS}, title = {An insight into the genome-wide analysis of bacterial defense mechanisms in a uropathogenic Morganella morganii isolate from Bangladesh.}, journal = {PloS one}, volume = {20}, number = {1}, pages = {e0313141}, doi = {10.1371/journal.pone.0313141}, pmid = {39847570}, issn = {1932-6203}, mesh = {*Morganella morganii/genetics/isolation & purification ; Bangladesh ; *Urinary Tract Infections/microbiology ; *Genome, Bacterial ; Humans ; *Phylogeny ; Genomic Islands/genetics ; Anti-Bacterial Agents/pharmacology ; }, abstract = {The gram-negative, facultative anaerobic bacterium Morganella morganii is linked to a number of illnesses, including nosocomial infections and urinary tract infections (UTIs). A clinical isolate from a UTI patient in Bangladesh was subjected to high-throughput whole genome sequencing and extensive bioinformatics analysis in order to gather knowledge about the genomic basis of bacterial defenses and pathogenicity in M. morganii. With an average nucleotide identity (ANI) of more than 97% similarity to a reference genome and phylogenetic analysis verified the isolate as M. morganii. Genome annotation identified 3,718 protein-coding sequences, including genes for metabolism, protein processing, stress response, energy, and membrane transport. The presence of biosynthetic gene clusters points to the isolate's ability to create bioactive compounds, including antibiotics. Genomic islands contained genes for metal transporters, stress proteins, toxin proteins, and genes related to horizontal gene transfer. The beta-lactam resistance gene blaDHA was found using antimicrobial resistance (AMR) gene analysis across three databases. The virulence genes kdsA and cheY, which may be involved in chemotaxis and lipopolysaccharide production, were also available in the isolate, suggesting its high pathogenicity. The genome contained mobile genetic components and defense mechanisms, such as restriction modification and CRISPR-Cas systems, indicating the bacterium's ability to defend itself against viral attacks. This thorough investigation sheds important light on M. morganii's pathogenicity and adaptive tactics by revealing its genetic characteristics, AMR, virulence components, and defense mechanisms. For the development of targeted treatments and preventing the onset of resistance in clinical care, it is essential to comprehend these genetic fingerprints.}, }
@article {pmid39847105, year = {2025}, author = {Karimi-Fard, A and Saidi, A and Tohidfar, M and Emami, SN}, title = {CRISPR-Cas-mediated adaptation of Thermus thermophilus HB8 to environmental stress conditions.}, journal = {Archives of microbiology}, volume = {207}, number = {2}, pages = {41}, pmid = {39847105}, issn = {1432-072X}, mesh = {*Thermus thermophilus/genetics/metabolism ; *CRISPR-Cas Systems ; *Stress, Physiological ; *Adaptation, Physiological/genetics ; *Bacterial Proteins/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; }, abstract = {Bacteria experience a continual array of environmental stresses, necessitating adaptive mechanisms crucial for their survival. Thermophilic bacteria, such as Thermus thermophilus, face constant environmental challenges, particularly high temperatures, which requires robust adaptive mechanisms for survival. Studying these extremophiles provides valuable insights into the intricate molecular and physiological processes used by extremophiles to adapt and survive in harsh environments. Through meta-analysis of microarray data, we revealed the key genes in T. thermophilus HB8 that respond to various environmental stresses. The analysis revealed 20 differentially expressed genes (DEGs), including 13 upregulated and seven downregulated genes, with a threshold of|log fold change| > 1 and an adjusted p-value < 0.05. Several genes identified as up-regulated in our analysis belonged to the CRISPR-associated protein (Cas) family. To validate these findings, we further evaluated the relative expression levels of TTHB188 (cas1/casA), TTHB189 (cas2/casB), TTHB190 (cas7/casC), TTHB191 (cas5/casD), TTHB192 (cas6/casE), and TTHB193 (cas1e) using RT-qPCR under H2O2 and salt stress conditions. The RT-qPCR analysis revealed significant up-regulation of transcripts, casA, casB, casC, casD, casE, and cas1e under salt stress. However, under H2O2 stress, only, casA, casB, and casC exhibited substantial increases in expression. Our findings may indicate that the CRISPR-associated proteins significantly impact the adaptive response of T. thermophilus HB8 to various environmental stresses, particularly salt stress, highlighting its significance in extremophile survival and adaptation. This research offers an important understanding of the complex strategies used by extremophiles to survive in challenging conditions.}, }
@article {pmid39845892, year = {2024}, author = {Hu, D and Hu, L and Lu, Y and Dong, X and Cao, X and Bai, S and Zhang, L and Li, D and Sun, Y}, title = {Intein-mediated split SaCas9 for genome editing in plants.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1506468}, pmid = {39845892}, issn = {2673-3439}, abstract = {Virus-induced genome editing (VIGE) technologies have been developed to address the limitations to plant genome editing, which heavily relies on genetic transformation and regeneration. However, the application of VIGE in plants is hampered by the challenge posed by the size of the commonly used gene editing nucleases, Cas9 and Cas12a. To overcome this challenge, we employed intein-mediated protein splicing to divide the SaCas9 transcript into two segments (Split-v1) and three segments (Split-v3). The Split-v1 system demonstrated genome editing efficiencies in transgenic plants comparable to those achieved with wild-type SaCas9, with efficiencies ranging from 70.2% to 96.1%. Additionally, we constructed barley stripe mosaic virus (BSMV)-based vectors to co-express Split-v1 SaCas9 and gRNAs targeting LcHRC, LcGW2, and LcTB1 in sheepgrass (Leymus chinensis), a Gramineae forage species known for its recalcitrance to genetic transformation. Infected leaves of sheepgrass exhibited genome editing efficiencies ranging from 10.40% to 37.03%. These results demonstrate the potential of intein-mediated split nuclease systems to broaden the applicability of VIGE in challenging plant species.}, }
@article {pmid39844455, year = {2025}, author = {Jonsdottir, TK and Paoletta, MS and Ishizaki, T and Hernandez, S and Ivanova, M and Herrera Curbelo, A and Saiki, PA and Selinger, M and Das, D and Henriksson, J and Bushell, ESC}, title = {A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkaf005}, pmid = {39844455}, issn = {1362-4962}, support = {2021-06602//Swedish Research Council/ ; 2019.0178//Knut and Alice Wallenberg Foundation/ ; 23 3102 Pj//Swedish Cancer Society/ ; 202160312//Japan Society for the Promotion of Science/ ; //Swedish Universities/ ; }, mesh = {*Plasmodium berghei/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Animals ; Malaria/parasitology ; Genome, Protozoan/genetics ; Genetic Vectors/genetics ; }, abstract = {Many Plasmodium genes remain uncharacterized due to low genetic tractability. Previous large-scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called P. berghei high-throughput (PbHiT), which uses a single cloning step to generate targeting vectors with 100-bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide an online resource of knockout and tagging designs to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasite's biology at scale.}, }
@article {pmid39844454, year = {2025}, author = {Garcia-Guerra, A and Sathyaprakash, C and de Jong, OG and Lim, WF and Vader, P and El Andaloussi, S and Bath, J and Reine, J and Aoki, Y and Turberfield, AJ and Wood, MJA and Rinaldi, C}, title = {Tissue-specific modulation of CRISPR activity by miRNA-sensing guide RNAs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkaf016}, pmid = {39844454}, issn = {1362-4962}, support = {MR/Y009703/1/MRC_/Medical Research Council/United Kingdom ; //Kennedy's Disease Association/ ; 578222//Muscular Dystrophy Association/ ; MR/N024850/1/MRC_/Medical Research Council/United Kingdom ; 317110//Marie Curie Initial Training Network EScoDNA/ ; //xford University Press John Fell Fund/ ; LCF/BQ/EU16/11560044//La Caixa Foundation Fellowship/ ; //University of Oxford/ ; }, mesh = {*MicroRNAs/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Animals ; *CRISPR-Cas Systems ; Argonaute Proteins/metabolism/genetics ; Mice ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; HEK293 Cells ; }, abstract = {Nucleic acid nanostructures offer unique opportunities for biomedical applications due to their sequence-programmable structures and functions, which enable the design of complex responses to molecular cues. Control of the biological activity of therapeutic cargoes based on endogenous molecular signatures holds the potential to overcome major hurdles in translational research: cell specificity and off-target effects. Endogenous microRNAs (miRNAs) can be used to profile cell type and cell state, and are ideal inputs for RNA nanodevices. Here, we present CRISPR MiRAGE (miRNA-activated genome editing), a tool comprising a dynamic single-guide RNA that senses miRNA complexed with Argonaute proteins and controls downstream CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) activity based on the detected miRNA signature. We study the operation of the miRNA-sensing single-guide RNA and attain muscle-specific activation of gene editing through CRISPR MiRAGE in models of Duchenne muscular dystrophy. By enabling RNA-controlled gene editing activity, this technology creates opportunities to advance tissue-specific CRISPR treatments for human diseases.}, }
@article {pmid39844394, year = {2025}, author = {Feng, S and Li, J and Yan, A and Zhu, X and Zhang, L and Tang, D and Liu, L}, title = {Application of Gene Editing in Triple-Negative Breast Cancer Research.}, journal = {Cell biochemistry and function}, volume = {43}, number = {1}, pages = {e70044}, doi = {10.1002/cbf.70044}, pmid = {39844394}, issn = {1099-0844}, support = {//This work was supported by the Scientific Research Project of Department of Education of Guangdong Province (Project Number:2023ZDZX2057) and GuangDong Basic and Applied Basic Research Foundation (Project Number:2023A1515140070)./ ; }, mesh = {Humans ; *Gene Editing ; *Triple Negative Breast Neoplasms/genetics/therapy ; Female ; Animals ; Precision Medicine ; }, abstract = {With the rapid development of gene editing technology, its application in breast cancer has gradually become the focus of research. This article reviews the application of gene editing technology in the treatment of breast cancer, and discusses its challenges and future development directions. The key application areas of gene editing technology in the treatment of breast cancer will be outlined, including the discovery of new therapeutic targets and the development of drugs related to the pathway. Gene editing technology has played an important role in the discovery of new therapeutic targets. Through the use of gene editing technology, breast cancer-related genes are systematically edited to regulate key regulatory factors on related pathways or key tumor suppressor genes such as FOXC1 and BRCA, and the results are analyzed in cell or animal experiments, and the target is obtained from the experimental results, which provides important clues for the development of new drugs. This approach provides an innovative way to find more effective treatment strategies and inhibit tumor growth. In addition, gene editing technology has also promoted the personalization of breast cancer treatment. By analyzing a patient's genomic information, researchers can pinpoint key genetic mutations in a patient's tumor and design personalized treatments. This personalized treatment approach is expected to improve the therapeutic effect and reduce adverse reactions. Finally, the application of gene editing technology also provides support for the development of breast cancer immunotherapy. By editing immune cells to make them more potent against tumors, researchers are trying to develop more effective immunotherapies to bring new treatment options to breast cancer patients.}, }
@article {pmid39843407, year = {2025}, author = {Liu, X and Huang, L and Ye, Y and Wang, H and Tang, M and He, F and Xia, Z and Deng, S and Zhang, P and Dai, R and Liang, S}, title = {Staphylococcus aureus nt5 gene mutation through CRISPR RNA-guided base editing weakens bacterial virulence and immune evasion.}, journal = {Virulence}, volume = {16}, number = {1}, pages = {2451163}, doi = {10.1080/21505594.2025.2451163}, pmid = {39843407}, issn = {2150-5608}, mesh = {*Staphylococcus aureus/genetics/pathogenicity/drug effects ; Animals ; Mice ; *Staphylococcal Infections/microbiology ; *Immune Evasion ; Virulence/genetics ; *5'-Nucleotidase/genetics ; *Mutation ; Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial/genetics ; Daptomycin/pharmacology ; Gene Editing/methods ; Female ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {The resistance of commonly used clinical antibiotics, such as daptomycin (DAP), has become increasingly serious in the fight against Staphylococcus aureus (S. aureus) infection. It is essential to explore key pathogenicity-driven genes/proteins in bacterial infection and antibiotics resistance, which contributes to develop novel therapeutic strategies against S. aureus infections. The nt5 gene of S. aureus, encoding 5'-nucleotidase (NT5), is nearly unknown for its function in drug resistance and bacterial infection. Herein, to reveal nt5 gene role in drug resistance and infection ability of S. aureus, we performed nt5[C166T] gene mutation using a clustered regulatory interspaced short palindromic repeat ribonucleic acid (RNA)-guided base editing system to investigate the lose-of-function of NT5 protein. Subsequent transcriptome sequencing of the mutant strain revealed that nt5 inactivation caused changes in cell membrane integrity and inhibited nucleotide metabolism, suggesting the nt5 gene may be involved in bacterial drug resistance and virulence. The mutant strain exhibited enhanced tolerance to DAP treatment by attenuating cell membrane potential dissipation and slowing deoxyribonucleic acid release. Moreover, the nt5 mutation alleviated abscess degree of mouse kidneys caused by S. aureus infection byreducing the expression of IL-1β, IL-6, and IL-18. The nt5 mutant strain was easily swallowed by host immune cells, resulting in weak bacterial toxicity of the S. aureus mutant in the bacterial infection process. In summary, nt5 gene mutation confers tolerance to DAP and a lower bacterial capacity to form kidney abscesses through phagocytosis of host immune cells, which indicates the targeted inhibition of NT5 protein would offer a potential new therapeutic strategy against S. aureus infection.}, }
@article {pmid39841730, year = {2025}, author = {Khan, H and Huang, X and Raj, V and Wang, H}, title = {A versatile site-directed gene trap strategy to manipulate gene activity and control gene expression in Caenorhabditis elegans.}, journal = {PLoS genetics}, volume = {21}, number = {1}, pages = {e1011541}, doi = {10.1371/journal.pgen.1011541}, pmid = {39841730}, issn = {1553-7404}, mesh = {*Caenorhabditis elegans/genetics ; Animals ; *CRISPR-Cas Systems ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gene Expression Regulation/genetics ; Animals, Genetically Modified ; Transgenes ; Transcription Factors/genetics/metabolism ; Gene Editing/methods ; }, abstract = {The ability to manipulate gene activity and control transgene expression is essential to study gene function. While several genetic tools for modifying genes or controlling expression separately are available for Caenorhabditis elegans, there are no genetic approaches to generate mutations that simultaneously disrupt gene function and provide genetic access to the cells expressing the disrupted gene. To achieve this, we developed a versatile gene trap strategy based on cGAL, a GAL4-UAS bipartite expression system for C. elegans. We designed a cGAL gene trap cassette and used CRISPR/Cas9 to insert it into the target gene, creating a bicistronic operon that simultaneously expresses a truncated endogenous protein and the cGAL driver in the cells expressing the target gene. We demonstrate that our cGAL gene trap strategy robustly generated loss-of-function alleles. Combining the cGAL gene trap lines with different UAS effector strains allowed us to rescue the loss-of-function phenotype, observe the gene expression pattern, and manipulate cell activity spatiotemporally. We show that, by recombinase-mediated cassette exchange (RMCE) via microinjection or genetic crossing, the cGAL gene trap lines can be further engineered in vivo to easily swap cGAL with other bipartite expression systems' drivers, including QF/QF2, Tet-On/Tet-Off, and LexA, to generate new gene trap lines with different drivers at the same genomic locus. These drivers can be combined with their corresponding effectors for orthogonal transgenic control. Thus, our cGAL-based gene trap is versatile and represents a powerful genetic tool for gene function analysis in C. elegans, which will ultimately provide new insights into how genes in the genome control the biology of an organism.}, }
@article {pmid39841239, year = {2025}, author = {Bohra, A and Tiwari, A and Pareek, S and Joshi, R and Satheesh Naik, SJ and Kumari, K and Verma, RL and Parihar, AK and Patil, PG and Dixit, GP}, title = {Past and future of cytoplasmic male sterility and heterosis breeding in crop plants.}, journal = {Plant cell reports}, volume = {44}, number = {2}, pages = {33}, pmid = {39841239}, issn = {1432-203X}, mesh = {*Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Plant Infertility/genetics ; *Hybrid Vigor/genetics ; *Pollen/genetics/physiology ; Gene Editing ; Cytoplasm/genetics ; }, abstract = {Plant breeding needs to embrace genetic innovations to ensure stability in crop yields under fluctuating climatic conditions. Development of commercial hybrid varieties has proven to be a sustainable and economical alternative to deliver superior yield, quality and resistance with uniformity in a number of food crops. Cytoplasmic male sterility (CMS), a maternally inherited inability to produce functional pollen, facilitates a three-line system for efficient hybrid seed production strategies in crops. The CMS system has illustrated its potential as a robust pollination control mechanism to support the billion-dollar seed industry. In plants, CMS arises due to a genomic conflict between mitochondrial open reading frames (orfs) and nuclear-encoding restoration-of-fertility (Rf) genes, leading to floral abnormalities and pollen sterility. Research on pollen sterility and fertility restoration provides deeper insights into cytoplasmic-nuclear interplay in plants and elucidates key molecular targets for hybrid breeding in crops. More recently, programmable gene editing (e.g., TALEN, CRISPR-Cas) has emerged as a promising tool to functionally validate CMS and Rf genes and obviate the need for pollen donors or Rf-genes for hybrid breeding. Modern genomic prediction models have allowed establishment of high-performing heterotic groups and patterns for sustaining long-term gain in hybrid breeding. This article reviews latest discoveries elucidating the molecular mechanisms behind CMS and fertility restoration in plants. We then present our perspective on how evolving genetic technologies are contributing to advance fundamental knowledge of the CMS-Rf genetic system for producing crop hybrids with high heterosis.}, }
@article {pmid39744795, year = {2025}, author = {Li, X and Huang, Z and Lau, CH and Li, J and Zou, M and Wu, W and Chen, X and Li, J and Huang, Y and Wang, T and Li, Y and Xu, M and Huang, X and Zhu, H and Yang, C}, title = {One-pot isothermal CRISPR/Dx system for specific and sensitive detection of microRNA.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {4}, pages = {823-833}, doi = {10.1039/d4ay01695e}, pmid = {39744795}, issn = {1759-9679}, mesh = {*MicroRNAs/genetics/analysis ; Humans ; *Pancreatic Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Sensitivity and Specificity ; }, abstract = {MicroRNA (miRNA) is a promising biomarker for the early diagnosis of pancreatic cancer. To enable sensitive and reliable miRNA detection, we have developed a one-pot isothermal CRISPR/Dx detection system by combining rolling circle amplification (RCA) and CRISPR/Cas12a. RCA and CRISPR/Cas12a reactions are carried out in a single closed tube, bypassing the transferring step. We demonstrate the feasibility of our one-pot CRISPR/Dx system in detecting pancreatic cancer by targeting miR-25, miR-191, miR-205, and miR-1246. When applied to fluorescence- and lateral flow strip paper-based detection platforms, our one-pot CRISPR/Dx system detects synthetic miR-25 at a LOD of 6.60 fM and 500 fM, respectively. It has high targeting specificity, as shown by its ability to discriminate miR-25 with a single-base mutation and highly homologous miRNA species. It is also successfully generalized to detect other pancreatic cancer-associated miRNAs, including miR-191, miR-205, and miR-1246. Importantly, our one-pot CRISPR/Dx system enables specific and sensitive detection of endogenous miR-25 in the human pancreatic cancer cell line PANC-1. We have successfully developed a one-pot isothermal CRISPR/Dx system for detecting miRNA with high specificity and sensitivity. It is highly flexible and economical, as a common crRNA can detect different miRNAs and only requires minor modifications to the locking padlock probe. Therefore, it can potentially be translated into clinical settings and POCT for the diagnosis of various human cancers.}, }
@article {pmid39732069, year = {2025}, author = {Zhang, J and Zhou, Y and Qiao, J and Liu, Y}, title = {Recent advances in spatiotemporal control of the CRISPR/Cas9 system.}, journal = {Colloids and surfaces. B, Biointerfaces}, volume = {248}, number = {}, pages = {114474}, doi = {10.1016/j.colsurfb.2024.114474}, pmid = {39732069}, issn = {1873-4367}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Animals ; }, abstract = {The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.}, }
@article {pmid39704119, year = {2025}, author = {Lv, L and Liu, S and Fu, Y and Zhang, Y and Wang, M and Sun, J and Wang, Y and Lu, Y and Niu, G}, title = {A tunable and reversible thermo-inducible bio-switch for streptomycetes.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1236}, pmid = {39704119}, issn = {1362-4962}, support = {2023YFD1700700//National Key Research and Development Program of China/ ; 32370077//National Natural Science Foundation of China/ ; }, mesh = {*Streptomyces/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; *Temperature ; Anti-Bacterial Agents/pharmacology/biosynthesis ; Bacterial Proteins/metabolism/genetics ; Anthraquinones/metabolism ; Gene Editing/methods ; Benzoisochromanequinones ; }, abstract = {Programmable control of bacterial gene expression holds great significance for both applied and academic research. This is particularly true for Streptomyces, a genus of Gram-positive bacteria and major producers of prodigious natural products. Despite that a few inducible regulatory systems have been developed for use in Streptomyces, there is an increasing pursuit to augment the toolkit of high-performance induction systems. We herein report a robust and reversible thermo-inducible bio-switch, designated as StrepT-switch. This bio-switch enables tunable and reversible control of gene expression using physiological temperatures as stimulation inputs. It has been proven successful in highly efficient CRISPR/Cas9-mediated genome engineering, as well as programmable control of antibiotic production and morphological differentiation. The versatility of the device is also demonstrated by thermal induction of a site-specific relaxase ZouA for overproduction of actinorhodin, a blue pigmented polyketide antibiotic. This study showcases the exploration a temperature-sensing module and exemplifies its versatility for programmable control of various target genes in Streptomyces species.}, }
@article {pmid39663454, year = {2025}, author = {Arce, MM and Umhoefer, JM and Arang, N and Kasinathan, S and Freimer, JW and Steinhart, Z and Shen, H and Pham, MTN and Ota, M and Wadhera, A and Dajani, R and Dorovskyi, D and Chen, YY and Liu, Q and Zhou, Y and Swaney, DL and Obernier, K and Shy, BR and Carnevale, J and Satpathy, AT and Krogan, NJ and Pritchard, JK and Marson, A}, title = {Central control of dynamic gene circuits governs T cell rest and activation.}, journal = {Nature}, volume = {637}, number = {8047}, pages = {930-939}, pmid = {39663454}, issn = {1476-4687}, mesh = {Humans ; *Lymphocyte Activation ; *Gene Regulatory Networks ; *CD4-Positive T-Lymphocytes/immunology/metabolism/cytology ; *Interleukin-2 Receptor alpha Subunit/metabolism/genetics ; *Single-Cell Analysis ; Kruppel-Like Transcription Factors/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Mediator Complex/metabolism/genetics ; Gene Expression Regulation ; T-Lymphocytes, Regulatory/immunology/metabolism/cytology ; }, abstract = {The ability of cells to maintain distinct identities and respond to transient environmental signals requires tightly controlled regulation of gene networks[1-3]. These dynamic regulatory circuits that respond to extracellular cues in primary human cells remain poorly defined. The need for context-dependent regulation is prominent in T cells, where distinct lineages must respond to diverse signals to mount effective immune responses and maintain homeostasis[4-8]. Here we performed CRISPR screens in multiple primary human CD4[+] T cell contexts to identify regulators that control expression of IL-2Rα, a canonical marker of T cell activation transiently expressed by pro-inflammatory effector T cells and constitutively expressed by anti-inflammatory regulatory T cells where it is required for fitness[9-11]. Approximately 90% of identified regulators of IL-2Rα had effects that varied across cell types and/or stimulation states, including a subset that even had opposite effects across conditions. Using single-cell transcriptomics after pooled perturbation of context-specific screen hits, we characterized specific factors as regulators of overall rest or activation and constructed state-specific regulatory networks. MED12 - a component of the Mediator complex - serves as a dynamic orchestrator of key regulators, controlling expression of distinct sets of regulators in different T cell contexts. Immunoprecipitation-mass spectrometry revealed that MED12 interacts with the histone methylating COMPASS complex. MED12 was required for histone methylation and expression of genes encoding key context-specific regulators, including the rest maintenance factor KLF2 and the versatile regulator MYC. CRISPR ablation of MED12 blunted the cell-state transitions between rest and activation and protected from activation-induced cell death. Overall, this work leverages CRISPR screens performed across conditions to define dynamic gene circuits required to establish resting and activated T cell states.}, }
@article {pmid39660638, year = {2025}, author = {Goh, CG and Bader, AS and Tran, TA and Belotserkovskaya, R and D'Alessandro, G and Jackson, SP}, title = {TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1163}, pmid = {39660638}, issn = {1362-4962}, support = {855741//ERC Synergy Award/ ; //A*STAR National Science Scholarship/ ; C17918/A28870//CRUK RadNet Cambridge Award/ ; 227014/Z/23/Z//Wellcome/ ; //University of Cambridge/ ; DRCPGM\100005/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {Humans ; *Phosphoric Diester Hydrolases/genetics/metabolism ; *Topoisomerase I Inhibitors/pharmacology ; *DNA Topoisomerases, Type I/genetics/metabolism ; *Camptothecin/pharmacology ; Mutation ; Cell Line ; RNA Splice Sites/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; }, abstract = {HAP1 is a near-haploid human cell line commonly used for mutagenesis and genome editing studies due to its hemizygous nature. We noticed an unusual hypersensitivity of HAP1 to camptothecin, an antineoplastic drug that stabilizes topoisomerase I cleavage complexes (TOP1ccs). We have attributed this hypersensitivity to a deficiency of TDP1, a key phosphodiesterase involved in resolving abortive TOP1ccs. Through whole-exome sequencing and subsequent restoration of TDP1 protein via CRISPR-Cas9 endogenous genome editing, we demonstrate that TDP1 deficiency and camptothecin hypersensitivity in HAP1 cells are a result of a splice-site mutation (TDP1 c.660-1G > A) that causes exon skipping and TDP1 loss of function. The lack of TDP1 in HAP1 cells should be considered when studying topoisomerase-associated DNA lesions and when generalizing mechanisms of DNA damage repair using HAP1 cells. Finally, we also report the generation of HAP1 STAR clones with restored TDP1 expression and function, which may be useful in further studies to probe cellular phenotypes relating to TOP1cc repair.}, }
@article {pmid39658047, year = {2025}, author = {Crawford, KD and Khan, AG and Lopez, SC and Goodarzi, H and Shipman, SL}, title = {High throughput variant libraries and machine learning yield design rules for retron gene editors.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1199}, pmid = {39658047}, issn = {1362-4962}, support = {MCB 2137692//National Science Foundation/ ; R21EB031393/EB/NIBIB NIH HHS/United States ; //W. M. Keck Foundation/ ; //Pew Biomedical Scholars Program/ ; //Gary and Eileen Morgenthaler Fund/ ; //UCSF Discovery Fellows Program/ ; R21EB031393/EB/NIBIB NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Machine Learning ; RNA-Directed DNA Polymerase/metabolism/genetics ; RNA, Untranslated/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The bacterial retron reverse transcriptase system has served as an intracellular factory for single-stranded DNA in many biotechnological applications. In these technologies, a natural retron non-coding RNA (ncRNA) is modified to encode a template for the production of custom DNA sequences by reverse transcription. The efficiency of reverse transcription is a major limiting step for retron technologies, but we lack systematic knowledge of how to improve or maintain reverse transcription efficiency while changing the retron sequence for custom DNA production. Here, we test thousands of different modifications to the Retron-Eco1 ncRNA and measure DNA production in pooled variant library experiments, identifying regions of the ncRNA that are tolerant and intolerant to modification. We apply this new information to a specific application: the use of the retron to produce a precise genome editing donor in combination with a CRISPR-Cas9 RNA-guided nuclease (an editron). We use high-throughput libraries in Saccharomyces cerevisiae to additionally define design rules for editrons. We extend our new knowledge of retron DNA production and editron design rules to human genome editing to achieve the highest efficiency Retron-Eco1 editrons to date.}, }
@article {pmid39657782, year = {2025}, author = {Li, Z and Wang, X and Janssen, JM and Liu, J and Tasca, F and Hoeben, RC and Gonçalves, MAFV}, title = {Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1213}, pmid = {39657782}, issn = {1362-4962}, support = {//China Scholarship Council/ ; //Prinses Beatrix Spierfonds/ ; //Dutch Duchenne Parent/ ; //Horizon Europe/ ; /NWO_/Dutch Research Council/Netherlands ; }, mesh = {*Genetic Vectors/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Dependovirus/genetics ; Humans ; HEK293 Cells ; Transgenes ; Adenoviridae/genetics ; DNA/genetics/metabolism ; Homologous Recombination ; DNA End-Joining Repair/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; }, abstract = {Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.}, }
@article {pmid39633028, year = {2025}, author = {Yin, JA and Frick, L and Scheidmann, MC and Liu, T and Trevisan, C and Dhingra, A and Spinelli, A and Wu, Y and Yao, L and Vena, DL and Knapp, B and Guo, J and De Cecco, E and Ging, K and Armani, A and Oakeley, EJ and Nigsch, F and Jenzer, J and Haegele, J and Pikusa, M and Täger, J and Rodriguez-Nieto, S and Bouris, V and Ribeiro, R and Baroni, F and Bedi, MS and Berry, S and Losa, M and Hornemann, S and Kampmann, M and Pelkmans, L and Hoepfner, D and Heutink, P and Aguzzi, A}, title = {Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {127-148}, pmid = {39633028}, issn = {2157-846X}, mesh = {Humans ; *Gene Silencing ; *CRISPR-Cas Systems/genetics ; *Genome, Human/genetics ; Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Gene Deletion ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcription Factors/genetics/metabolism ; }, abstract = {Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrP[C], screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.}, }
@article {pmid39496933, year = {2025}, author = {Hwang, GH and Lee, SH and Oh, M and Kim, S and Habib, O and Jang, HK and Kim, HS and Kim, Y and Kim, CH and Kim, S and Bae, S}, title = {Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {79-92}, pmid = {39496933}, issn = {2157-846X}, support = {2021M3A9H3015389//National Research Foundation of Korea (NRF)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *DNA Repair/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; Sequence Deletion/genetics ; DNA/genetics ; Cell Line, Tumor ; DNA End-Joining Repair/genetics ; T-Lymphocytes ; Endonucleases/genetics/metabolism ; }, abstract = {When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.}, }
@article {pmid39428427, year = {2025}, author = {Yang, F and Wei, N and Cai, S and Liu, J and Lan, Q and Zhang, H and Shang, L and Zheng, B and Wang, M and Liu, Y and Zhang, L and Fei, C and Tong, W and Liu, C and Kuang, E and Tong, G and Gu, F}, title = {Genome-wide CRISPR screens identify CLC-2 as a drug target for anti-herpesvirus therapy: tackling herpesvirus drug resistance.}, journal = {Science China. Life sciences}, volume = {68}, number = {2}, pages = {515-526}, pmid = {39428427}, issn = {1869-1889}, mesh = {*Chloride Channels/genetics/metabolism ; Animals ; *Antiviral Agents/pharmacology ; Mice ; *CLC-2 Chloride Channels ; *Drug Resistance, Viral/genetics ; *Herpesvirus 1, Suid/drug effects/genetics ; Humans ; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; Herpesviridae/drug effects/genetics ; Virus Replication/drug effects ; }, abstract = {The emergence of drug resistance to virus (i.e., acyclovir (ACV) to herpesviruses) has been termed one of the common clinical issues, emphasizing the discovery of new antiviral agents. To address it, a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening was performed in mouse haploid embryonic stem cells infected with pseudorabies virus (PRV), an α-herpesvirus causing human and pig diseases. The results demonstrated that type 2 voltage-gated chloride channels (CLC-2) encoded by one of the identified genes, CLCN2, is a potential drug target for anti-herpesvirus therapy. CLC-2 inhibitors, omeprazole (OME) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), can efficiently inhibit infection of multiple herpesviruses in cellulo (i.e., PRV, HSV and EBV), and effectively treat murine herpes simplex encephalitis (HSE). Additionally, DIDS was found to inhibit HSV-1 replication by blocking the PI3K/Akt pathway. Most importantly, both DIDS and OME were able to inhibit ACV-resistant HSV-1 strain infection. The study's findings suggest that targeting host-cell factors such as CLC-2 may be a promising approach to tackling herpesvirus drug resistance. The discovery of CLC-2 as a potential drug target for anti-herpesvirus therapy provides a new direction for the development of novel antiviral agents.}, }
@article {pmid39265628, year = {2025}, author = {Crissey, MAS and Versace, A and Bhardwaj, M and Jain, V and Liu, S and Singh, A and Beer, LA and Tang, HY and Villanueva, J and Gimotty, PA and Xu, X and Amaravadi, RK}, title = {Divergent effects of acute and chronic PPT1 inhibition in melanoma.}, journal = {Autophagy}, volume = {21}, number = {2}, pages = {394-406}, doi = {10.1080/15548627.2024.2403152}, pmid = {39265628}, issn = {1554-8635}, support = {P01 CA114046/CA/NCI NIH HHS/United States ; P30 CA010815/CA/NCI NIH HHS/United States ; P50 CA261608/CA/NCI NIH HHS/United States ; R01 CA266404/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Melanoma/genetics/pathology/drug therapy/metabolism ; Humans ; Mice ; *Autophagy/drug effects/physiology/genetics ; *Thiolester Hydrolases/metabolism/genetics/antagonists & inhibitors ; *Mice, Knockout ; Proto-Oncogene Mas ; Cell Line, Tumor ; Lysosomes/metabolism/drug effects ; CRISPR-Cas Systems/genetics ; }, abstract = {Macroautophagy/autophagy-lysosome function promotes growth and survival of cancer cells, making them attractive targets for cancer therapy. One intriguing lysosomal target is PPT1 (palmitoyl-protein thioesterase 1). PPT1 inhibitors derived from chloroquine block autophagy, have significant antitumor activity in preclinical models and are being developed for clinical trials. However, the role of PPT1 in tumorigenesis remains poorly understood. Here we report that in melanoma cells, acute siRNA or pharmacological PPT1 inhibition led to increased ferroptosis sensitivity and significant loss of viability, whereas chronic PPT1 knockout using CRISPR-Cas9 produced blunted ferroptosis that led to sustained viability and growth. Each mode of PPT1 inhibition produced lysosome-autophagy inhibition but distinct proteomic changes, demonstrating the complexity of cellular adaptation mechanisms. To determine whether total genetic loss of Ppt1 would affect tumorigenesis in vivo, we developed a Ppt1 conditional knockout mouse model. We then crossed it into the BrafCA, PtenloxP, Tyr:CreERT2 melanoma mouse model to investigate the impact of Ppt1 loss on tumorigenesis. Loss of Ppt1 had no impact on melanoma histology, time to tumor initiation, or survival of tumor-bearing mice. These results suggest that chemical PPT1 inhibitors produce different adaptations than genetic PPT1 inhibition, and additional studies are warranted to fully understand the mechanism of chloroquine derivatives that target PPT1 in cancer.Abbreviations: 4-HT: 4-hydroxytamoxifen; BRAF: B-Raf proto-oncogene, serine/threonine kinase; cKO: conditional knockout; CRISPR-Cas9: clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9; DC661: A specific PPT1 inhibitor; DMSO: dimethyl sulfoxide; Dox; doxycycline hyclate; Easi-CRISPR: efficient additions with ssDNA inserts-CRISPR; GNS561/ezurpimtrostat: A PPT1 inhibitor; Hug: human guide; iCas: inducible CRISPR-Cas9; KO: knockout; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; LDLR: low density lipoprotein receptor; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NT: non-target; PTEN: phosphatase and tensin homolog; PPT1: palmitoyl-protein thioesterase 1; RSL3: RAS-selective lethal small molecule 3; SCRIB/SCRB1: scribble planar cell polarity protein; Tyr:CreERT2: tyrosinase-driven Cre recombinase fused with the tamoxifen-inducible mutant ligand binding domain of the human estrogen receptor; UGCG: UDP-glucose ceramide glucosyltransferase; WT: wild-type.}, }
@article {pmid39187662, year = {2025}, author = {Tong, M and Palmer, N and Dailamy, A and Kumar, A and Khaliq, H and Han, S and Finburgh, E and Wing, M and Hong, C and Xiang, Y and Miyasaki, K and Portell, A and Rainaldi, J and Suhardjo, A and Nourreddine, S and Chew, WL and Kwon, EJ and Mali, P}, title = {Robust genome and cell engineering via in vitro and in situ circularized RNAs.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {109-126}, pmid = {39187662}, issn = {2157-846X}, support = {W81XWH-22-1-0401//U.S. Department of Defense (United States Department of Defense)/ ; R01 NS131560/NS/NINDS NIH HHS/United States ; AHA 916973//American Heart Association (American Heart Association, Inc.)/ ; U54CA274502//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; U54 CA274502/CA/NCI NIH HHS/United States ; DP2NS111507//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; OT2OD032742//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; }, mesh = {*RNA, Circular/genetics ; *Cell Engineering/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Myocytes, Cardiac/metabolism/cytology ; Neurons/metabolism ; Mice ; HEK293 Cells ; Genome/genetics ; RNA/genetics ; Introns/genetics ; Gene Editing/methods ; Protein Engineering/methods ; }, abstract = {Circularization can improve RNA persistence, yet simple and scalable approaches to achieve this are lacking. Here we report two methods that facilitate the pursuit of circular RNAs (cRNAs): cRNAs developed via in vitro circularization using group II introns, and cRNAs developed via in-cell circularization by the ubiquitously expressed RtcB protein. We also report simple purification protocols that enable high cRNA yields (40-75%) while maintaining low immune responses. These methods and protocols facilitate a broad range of applications in stem cell engineering as well as robust genome and epigenome targeting via zinc finger proteins and CRISPR-Cas9. Notably, cRNAs bearing the encephalomyocarditis internal ribosome entry enabled robust expression and persistence compared with linear capped RNAs in cardiomyocytes and neurons, which highlights the utility of cRNAs in these non-dividing cells. We also describe genome targeting via deimmunized Cas9 delivered as cRNA and a long-range multiplexed protein engineering methodology for the combinatorial screening of deimmunized protein variants that enables compatibility between persistence of expression and immunogenicity in cRNA-delivered proteins. The cRNA toolset will aid research and the development of therapeutics.}, }
@article {pmid38987629, year = {2025}, author = {Sousa, AA and Hemez, C and Lei, L and Traore, S and Kulhankova, K and Newby, GA and Doman, JL and Oye, K and Pandey, S and Karp, PH and McCray, PB and Liu, DR}, title = {Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells.}, journal = {Nature biomedical engineering}, volume = {9}, number = {1}, pages = {7-21}, pmid = {38987629}, issn = {2157-846X}, support = {P01HL152960//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; R35GM118062//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; T32 GM008313/GM/NIGMS NIH HHS/United States ; UH3 HL147366/HL/NHLBI NIH HHS/United States ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; }, mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Gene Editing/methods ; *Epithelial Cells/metabolism ; HEK293 Cells ; *Cystic Fibrosis/genetics/therapy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bronchi/cytology ; }, abstract = {Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE-engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal 'dead' single-guide RNAs-we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.}, }
@article {pmid39840369, year = {2024}, author = {Moss, O and Li, X and Wang, ES and Kanagarajan, S and Guan, R and Ivarson, E and Zhu, LH}, title = {Knockout of BnaX.SGT.a caused significant sinapine reduction in transgene-free rapeseed mutants generated by protoplast-based CRISPR RNP editing.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1526941}, pmid = {39840369}, issn = {1664-462X}, abstract = {Rapeseed (Brassica napus L.) is known for its high-quality seed oil and protein content. However, its use in animal feed is restricted due to antinutritional factors present in the seedcake, with sinapine being one of the main compounds that reduces palatability. Attempts to develop rapeseed germplasm with lower sinapine levels through traditional breeding methods have shown limited progress. Genetic transformation methods could create new genotypes with reduced sinapine levels by silencing key genes involved in sinapine biosynthesis, though these methods often result in transgenic or genetically modified plants. The recent development of CRISPR-Cas technology provides a precise and efficient approach to crop improvement, with the potential to generate transgene-free mutants. In this study, we targeted the BnaX.SGT.a genes for knockout using CRISPR-Cas editing. By utilizing our newly established protoplast regeneration and transfection protocol for rapeseed, we demonstrated that DNA-free CRISPR editing via protoplast-based ribonucleoprotein (RNP) delivery was highly effective. We achieved successful knockout of the BnaX.SGT.a paralogues, with an average mutation efficiency of over 30%. Sequencing results revealed a variety of mutation types, from 1 bp insertions to 10 bp deletions, with most mutants exhibiting frameshift mutations that led to premature stop codons. The mutants displayed no visible phenotypic differences in growth patterns or flowering compared to the wild type. Importantly, sinapine content was significantly reduced in all T2 generation mutants analysed, while seed weight remained comparable between mutants and the wild type.}, }
@article {pmid39838465, year = {2025}, author = {Cheng, J and Chen, J and Chen, D and Li, B and Wei, C and Liu, T and Wang, X and Wen, Z and Jin, Y and Sun, C and Yang, G}, title = {Development of a Komagataella phaffii cell factory for sustainable production of (+)-valencene.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {29}, pmid = {39838465}, issn = {1475-2859}, support = {2024SSYS0103//the major science and technology project of Zhejiang Province/ ; 2023C4S02002//the Start-up funds of Xianghu Laboratory/ ; }, mesh = {*Sesquiterpenes/metabolism ; *Metabolic Engineering/methods ; Saccharomycetales/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Sesquiterpene (+)-valencene is a characteristic aroma component from sweet orange fruit, which has a variety of biological activities and is widely used in industrial manufacturing of food, beverage and cosmetics industries. However, at present, the content in plant sources is low, and its yield and quality would be influenced by weather and land, which limit the supply of (+)-valencene. The rapid development of synthetic biology has accelerated the construction of microbial cell factories and provided an effective alternative method for the production of natural products.
RESULTS: In this study, we first introduced the ( +)-valencene synthase into Komagataella phaffii by CRISPR/Cas9 system, and successfully constructed a ( +)-valencene producer with the initial yield of 2.1 mg/L. Subsequently, the ( +)-valencene yield was increased to 8.2 mg/L by fusing farnesyl pyrophosphate synthase with ( +)-valencene synthase using the selected ligation linker. High expression of key genes IDI1, tHMG1, ERG12 and ERG19 enhanced metabolic flux of MVA pathway, and the yield of ( +)-valencene was further increased by 27%. Besides, in-situ deletion of the promoter of ERG9 increased the yield of ( +)-valencene to 48.1 mg/L. Finally, we optimized the copy number of farnesyl pyrophosphate synthase and ( +)-valencene synthase fusion protein, and when the copy number reached three, the yield of ( +)-valencene achieved 173.6 mg/L in shake flask level, which was 82-fold higher than that of the starting strain CaVAL1.
CONCLUSIONS: The results obtained here suggest that K. phaffii has the potential to efficiently synthesize other terpenoids.}, }
@article {pmid39838422, year = {2025}, author = {Hou, S and Yang, S and Bai, W}, title = {Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.}, journal = {Microbial cell factories}, volume = {24}, number = {1}, pages = {28}, pmid = {39838422}, issn = {1475-2859}, support = {NO. XDC0120203//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; No. 306GJHZ2022010MI//CAS-NSTDA Joint Research Program/ ; TSBICIP-KJGG-020//Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Homologous Recombination ; Genome, Fungal ; }, abstract = {BACKGROUND: Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved.
RESULTS: In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha.
CONCLUSIONS: This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.}, }
@article {pmid39837388, year = {2025}, author = {Wang, X and Li, Y and Friess, D and Yao, L and Wang, X and He, Z and He, W and Li, M and Wang, W}, title = {Guanidyl-rich highly branched poly(β-amino ester)s for the delivery of dual CRISPR ribonucleoprotein for efficient large DNA fragment deletion.}, journal = {Journal of controlled release : official journal of the Controlled Release Society}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.jconrel.2025.01.032}, pmid = {39837388}, issn = {1873-4995}, abstract = {Gene editing technologies, particularly clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, have revolutionized the ability to modify gene sequences in living cells for therapeutic purposes. Delivery of CRISPR/Cas ribonucleoprotein (RNP) is preferred over its DNA and RNA formats in terms of gene editing effectiveness and low risk of off-target events. However, the intracellular delivery of RNP poses significant challenges and necessitates the development of non-viral vectors. Our previous study has demonstrated that phenyl guanidine (PG) group modified linear poly(β-amino ester)s (PAEs) can facilitate CRISPR/Cas9 RNP mediated gene knockout in HeLa cells. Here, we further investigated the utilization of highly branched PAEs (HPAEs) with PG groups (HPAE-PG) for efficient delivery of cytosolic protein and CRISPR/Cas9 RNP complexes, while also examining the influence of branching units and branching ratios on the delivery process. The efficiency of HPAE-PG/RNP transfection for large DNA fragment deletion was assessed using a dual sgRNA-guided approach to delete exon 80 of the human COL7A1 gene, which harbors mutations associated with dystrophic epidermolysis bullosa (DEB). Our findings demonstrate that HPAE-PG/RNP successfully induced a deletion of 56 base pairs (exon 80) within COL7A1 in both HEK cells and keratinocytes derived from recessive DEB patients. This study highlights the potential of HPAE-PG as a non-viral vector for large DNA fragment deletion, emphasizing the importance of branching factors of HPAEs in optimizing CRISPR RNP delivery for therapeutic applications in genetic disorders.}, }
@article {pmid39791925, year = {2025}, author = {Guo, W and Guo, Y and Xu, H and Li, C and Zhang, X and Zou, X and Sun, Z}, title = {Ultrasensitive "On-Off" Ratiometric Fluorescence Biosensor Based on RPA-CRISPR/Cas12a for Detection of Staphylococcus aureus.}, journal = {Journal of agricultural and food chemistry}, volume = {73}, number = {3}, pages = {2167-2173}, doi = {10.1021/acs.jafc.4c12202}, pmid = {39791925}, issn = {1520-5118}, mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Fluorescence ; Bacterial Proteins/genetics/metabolism ; Food Contamination/analysis ; Limit of Detection ; CRISPR-Associated Proteins/genetics/metabolism ; Recombinases/metabolism/genetics ; Metal-Organic Frameworks/chemistry ; Endodeoxyribonucleases/genetics/metabolism ; }, abstract = {Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal-organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 10[0] to 7.9 × 10[8] CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.}, }
@article {pmid39663988, year = {2025}, author = {Xiao, X and Zhang, C and Zhang, L and Zuo, C and Wu, W and Cheng, F and Wu, D and Xie, G and Mao, X and Yang, Y}, title = {A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection.}, journal = {Journal of materials chemistry. B}, volume = {13}, number = {4}, pages = {1372-1382}, doi = {10.1039/d4tb02178a}, pmid = {39663988}, issn = {2050-7518}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Bacteriophages/genetics ; Urinary Tract Infections/diagnosis/microbiology ; Escherichia coli/isolation & purification/genetics ; Bacteria/isolation & purification/genetics/virology ; }, abstract = {Rapid and accurate detection of viable bacteria is essential for the clinical diagnosis of urinary tract infections (UTIs) and for making effective therapeutic decisions. However, most current molecular diagnostic techniques are unable to differentiate between viable and non-viable bacteria. In this study, we introduce a novel isothermal platform that integrates strand exchange amplification (SEA) with the CRISPR/Cas12a system, thereby enhancing both the sensitivity and specificity of the assay and achieving detection of phage DNA at concentrations as low as 4 × 10[2] copies per μL. Moreover, the incorporation of phages facilitates the specific recognition of viable bacteria and amplifies the initial signal through the inherent specificity and propagation properties of these phages. By employing the phage-assisted SEA-Cas12a approach, we successfully detected viable bacteria in human urine samples without the necessity of DNA extraction within 3.5 hours, achieving a detection limit of 10[3] CFU per mL. Considering its speed, accuracy, and independence from specialized equipment, this platform demonstrates significant potential as a robust tool for the rapid detection of various pathogens in resource-limited settings, thereby facilitating timely clinical management of UTI patients.}, }
@article {pmid39377274, year = {2025}, author = {Tonsager, AJ and Stargell, LA}, title = {An undergraduate research experience in CRISPR-Cas9 mediated eukaryotic genome editing to teach fundamental biochemistry techniques.}, journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology}, volume = {53}, number = {1}, pages = {33-45}, doi = {10.1002/bmb.21862}, pmid = {39377274}, issn = {1539-3429}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Students ; Humans ; *Saccharomyces cerevisiae/genetics ; *Biochemistry/education ; Universities ; Research/education ; Curriculum ; }, abstract = {CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings.}, }
@article {pmid39039307, year = {2024}, author = {Ferreira da Silva, J and Tou, CJ and King, EM and Eller, ML and Rufino-Ramos, D and Ma, L and Cromwell, CR and Metovic, J and Benning, FMC and Chao, LH and Eichler, FS and Kleinstiver, BP}, title = {Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases.}, journal = {Nature biotechnology}, volume = {}, number = {}, pages = {}, pmid = {39039307}, issn = {1546-1696}, support = {R35GM142553//U.S. Department of Health Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM142553/GM/NIGMS NIH HHS/United States ; UM1-HG012010//U.S. Department of Health Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P180777//Swiss National Science Foundation | National Center of Competence in Research Quantum Science and Technology (NCCR QSIT - Quantum Science and Technology)/ ; DP2-CA281401//U.S. Department of Health Human Services | NIH | NIH Office of the Director (OD)/ ; ECOR FMD Fellowship//Massachusetts General Hospital (MGH)/ ; Kayden-Lambert MGH Research Scholar Award 2023-2028//Massachusetts General Hospital (MGH)/ ; P01-HL142494//U.S. Department of Health Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; FARA Fellowship//Friedreich's Ataxia Research Alliance (FARA)/ ; ALTF 750-2022//European Molecular Biology Organization (EMBO)/ ; 2020295403//National Science Foundation (NSF)/ ; U54 NS115052/NS/NINDS NIH HHS/United States ; Howard M. Goodman Fellowship//Massachusetts General Hospital (MGH)/ ; P01 HL142494/HL/NHLBI NIH HHS/United States ; UM1 HG012010/HG/NHGRI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; }, abstract = {Genome editing technologies based on DNA-dependent polymerases (DDPs) could offer several benefits compared with other types of editors to install diverse edits. Here, we develop click editing, a genome writing platform that couples the advantageous properties of DDPs with RNA-programmable nickases to permit the installation of a range of edits, including substitutions, insertions and deletions. Click editors (CEs) leverage the 'click'-like bioconjugation ability of HUH endonucleases with single-stranded DNA substrates to covalently tether 'click DNA' (clkDNA) templates encoding user-specifiable edits at targeted genomic loci. Through iterative optimization of the modular components of CEs and their clkDNAs, we demonstrate the ability to install precise genome edits with minimal indels in diverse immortalized human cell types and primary fibroblasts with precise editing efficiencies of up to ~30%. Editing efficiency can be improved by rapidly screening clkDNA oligonucleotides with various modifications, including repair-evading substitutions. Click editing is a precise and versatile genome editing approach for diverse biological applications.}, }
@article {pmid37884392, year = {2023}, author = {Liu, J and Lu, J and Yao, B and Zhang, Y and Huang, S and Chen, X and Shen, Y and Wang, X}, title = {Construction of humanized CYP1A2 rats using CRISPR/Cas9 to promote drug metabolism and pharmacokinetic research.}, journal = {Drug metabolism and disposition: the biological fate of chemicals}, volume = {52}, number = {1}, pages = {}, doi = {10.1124/dmd.123.001500}, pmid = {37884392}, issn = {1521-009X}, mesh = {Animals ; *Cytochrome P-450 CYP1A2/genetics/metabolism ; Humans ; Rats ; *CRISPR-Cas Systems ; *Microsomes, Liver/metabolism ; Male ; Clozapine/analogs & derivatives/pharmacokinetics/metabolism ; Gene Editing/methods ; Rats, Sprague-Dawley ; Theophylline/pharmacokinetics/metabolism/analogs & derivatives ; }, abstract = {Cytochrome P450 family 1 subfamily A member 2 (CYP1A2), performs an indispensable role in metabolism of both exogenous and endogenous substances. What is more, CYP1A2 functions in human diseases by regulating homeostasis of cholesterol. Despite the emergence of gene-editing animal models, genetically humanized animals that overcome species differences for further exploring the role of CYP1A2 in drug metabolism and human diseases have not yet been constructed. In this study, we inserted human CYP1A2 cDNA into the rat Cyp1a2 gene by using CRISPR/Cas9 technology. Results showed that human CYP1A2 was successfully expressed in humanized rat liver and there were no statistically significant differences of physiological symptoms compared with wild-type (WT) rats. In vitro incubation results indicated the different inhibition of furafylline on CYP1A2 activity in human liver microsomes, humanized CYP1A2 (hCYP1A2) rat liver microsomes, and WT rat liver microsomes, with IC50 values of 7.1 μM, 36.5 μM, and 285.8 μM, respectively. Meanwhile, pharmacokinetic characteristics of clozapine were conducted, and the results suggested that in hCYP1A2 rats, clozapine tended to be metabolized into norclozapine. Both the in vitro and in vivo results demonstrated the different metabolic functions of CYP1A2 in humanized and WT rats. We successfully constructed a novel humanized CYP1A2 rat model using the CRISPR/Cas9 system, providing a powerful tool for better predicting CYP1A2-mediated drug metabolism and pharmacokinetics. Significance Statement Human CYP1A2 takes active part both in the biotransformation of exogenous substances and endogenous substances. Meanwhile, it plays a regulatory role in human diseases, including hypercholesterolemia, hypertension as well as various malignant tumors. This study successfully constructed humanized CYP1A2 rat model by CRISPR/Cas9 technology, providing a powerful model for promoting drug development and safety evaluation, as well as further exploring the role of CYP1A2 in human diseases.}, }
@article {pmid39836103, year = {2025}, author = {Mai, Z and Zhou, T and Lin, Z}, title = {Detecting CYP2C19 genes through an integrated CRISPR/Cas13a-assisted system.}, journal = {Analytical methods : advancing methods and applications}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4ay01930j}, pmid = {39836103}, issn = {1759-9679}, abstract = {CYP2C19 gene single nucleotide polymorphisms (SNPs) should be considered in the clinical use of clopidogrel as they have important guiding value for predicting the risk of bleeding and thrombosis after clopidogrel treatment. The CRISPR/Cas system is increasingly used for SNP detection owing to its single-nucleotide mismatch specificity. Simultaneous detection of multiple SNPs for rapid identification of the CYP2C19 genotype is important, but there is no method to detect a wide variety of CYP2C19 SNPs. This study proposes a new integrated system that integrates the PCR reaction and CRISPR/Cas detection of three CYP2C19 genes on a device, achieving rapid, sensitive, and specific detection. In our design, magnetic beads with three different sizes capture target nucleic acid from the sample, which are dragged through different areas by magnetic force, for PCR amplification reaction and CRISPR/Cas13a detection of CYP2C19*2, CYP2C19*3 and CYP2C19*17 genes. Note that magnetic beads were sorted via microporous PC membranes of different apertures. This study exhibits a broad clinical application prospect and provides a favorable tool for clinical clopidogrel administration.}, }
@article {pmid39834176, year = {2025}, author = {Tang, M}, title = {Research Status of Clustered Regulary Interspaced Short Palindromic Repeats Technology in the Treatment of Human Papillomavirus (HPV) Infection Related Diseases.}, journal = {Cancer control : journal of the Moffitt Cancer Center}, volume = {32}, number = {}, pages = {10732748241300654}, doi = {10.1177/10732748241300654}, pmid = {39834176}, issn = {1526-2359}, mesh = {Humans ; *Papillomavirus Infections/therapy/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Female ; Papillomaviridae/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Uterine Cervical Neoplasms/therapy/virology/genetics ; Gene Editing/methods ; Human Papillomavirus Viruses ; }, abstract = {Background: CRISPR/Cas9 technology has rapidly advanced as a pivotal tool in cancer research, particularly in the precision targeting required for both detecting and treating malignancies. Its high specificity and low off-target effects make it exceptionally effective in applications involving Human Papillomavirus (HPV) related diseases, most notably cervical cancer. This approach offers a refined methodology for the rapid detection of viral infections and provides a robust platform for the safe and effective treatment of diseases associated with viral infections through gene therapy.Purpose: Gene therapy, within this context, involves the strategic delivery of genetic material into target cells via a vector. This is followed by the meticulous modulation of gene expression, whether through correction, addition, or suppression, specifically honed to target tumor cells while sparing healthy cells. This dual capacity to diagnose and treat at such a precise level underscores the transformative potential of CRISPR/Cas9 in contemporary medical science, particularly in oncology and virology.Research Design: This article provides an overview of the advancements made in utilizing the CRISPR-Cas9 system as a research tool for HPV-related treatments while summarizing its application status in basic research, diagnosis, and treatment of HPV.Data Collection: Furthermore, it discusses the future prospects for this technology within emerging areas of HPV research and precision medicine in clinical practice, while highlighting technical challenges and potential directions for future development.}, }
@article {pmid39833630, year = {2025}, author = {Zhang, B and Li, J and Yu, W}, title = {Integration of CRISPR/dCas9-Based methylation editing with guide positioning sequencing identifies dynamic changes of mrDEGs in breast cancer progression.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {82}, number = {1}, pages = {46}, pmid = {39833630}, issn = {1420-9071}, support = {32270645//National Natural Science Foundation of China/ ; 18JC1411101//Major Special Projects of Basic Research of Shanghai Science and Technology Commission/ ; }, mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *DNA Methylation/genetics ; Female ; *CRISPR-Cas Systems/genetics ; *Disease Progression ; *Gene Editing/methods ; *Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Dynamic changes in DNA methylation are prevalent during the progression of breast cancer. However, critical alterations in aberrant methylation and gene expression patterns have not been thoroughly characterized. Here, we utilized guide positioning sequencing (GPS) to conduct whole-genome DNA methylation analysis in a unique human breast cancer progression model: MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and metastatic carcinoma). By integrating with mRNA-seq and matched clinical expression data from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), six representative methylation-related differentially expressed genes (mrDEGs) were identified, including CAVIN2, ARL4D, DUSP1, TENT5B, P3H2, and MMP28. To validate our findings, we independently developed and optimized the dCas9-DNMT3L-DNMT3A system, achieving a high efficiency with a 98% increase in methylation at specific sites. DNA methylation levels significantly increased for the six genes, with CAVIN2 at 67.75 ± 1.05%, ARL4D at 53.29 ± 6.32%, DUSP1 at 57.63 ± 8.46%, TENT5B at 44.00 ± 5.09%, P3H2 at 58.50 ± 3.90%, and MMP28 at 49.60 ± 5.84%. RT-qPCR confirmed an inverse correlation between increased DNA methylation and gene expression. Most importantly, we mimicked tumor progression in vitro, demonstrating that transcriptional silencing of the TENT5B promotes cell proliferation in MCF10A cells owing to the crosstalk between hypermethylation and histone deacetylation. This study unveils the practical implications of DNA methylation dynamics of mrDEGs in reshaping epigenomic features during breast cancer malignant progression through integrated data analysis of the methylome and transcriptome. The application of the CRISPR/dCas9-based methylation editing technique elucidates the regulatory mechanisms and functional roles of individual genes within the DNA methylation signature, providing valuable insights for understanding breast cancer pathogenesis and facilitating potential therapeutic approaches in epigenome editing for patients with breast cancer.}, }
@article {pmid39833501, year = {2025}, author = {Gawlig, C and Hirschberger, R and Hanci, G and Schott, S and Marandi, S and Hesse, IR and Rühl, M}, title = {Full sequencing of 100mer sgRNA via tandem mass spectrometry by targeted RNase H digestion with customized probes.}, journal = {Analytical and bioanalytical chemistry}, volume = {}, number = {}, pages = {}, pmid = {39833501}, issn = {1618-2650}, abstract = {The use of single-guide RNA (sgRNA) for gene editing using the CRISPR Cas9 system has become a powerful technique in various fields, especially with the growing interest in such molecules as therapeutic options in the last years. An important parameter for the use of these molecules is the verification of the correct sgRNA oligonucleotide sequence. Apart from next-generation sequencing protocols, mass spectrometry (MS) has been proven as a powerful technique for this purpose. The protocol and investigations presented in this work show an optimal digestion and 100% sequence coverage of sgRNA, while top-down approaches or other ribonuclease (RNase) digestion strategies obtain a sequence coverage of up to 80-90% utilizing multiple RNases. The results in this publication were obtained by utilizing DNA-RNA hybrid GAPmer-like probes and RNase H, an enzyme which specifically hydrolyzes RNA in DNA-RNA double strands. We assessed the optimal length of the DNA segment of these hybrid probes to maximize the specificity of the RNase H digestion and to achieve complete sequence confirmation by tandem MS analysis of the resulting digestion products. Furthermore, we showed that the approach is applicable for the identification of common synthesis-related impurities, like truncations and elongations. Despite the fact that the accessibility of this approach for highly modified molecules is limited to nucleotides which are not 2'-O-methylated, the optimized sequence coverage makes it a viable method.}, }
@article {pmid39833409, year = {2025}, author = {Zhang, X and Qiu, M and Han, B and Liao, L and Peng, X and Lin, J and Zhang, N and Hai, L and Liang, L and Ma, Y and Li, W and Liu, M}, title = {Generation and propagation of high fecundity gene edited fine wool sheep by CRISPR/Cas9.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2557}, pmid = {39833409}, issn = {2045-2322}, support = {2023SNGGGCC010//the Tianshan Talent Training Program-high-level talents for the advancement of modern agriculture and animal husbandry./ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region./ ; }, mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Fertility/genetics ; Sheep/genetics ; Female ; Wool ; Bone Morphogenetic Protein Receptors, Type I/genetics ; Litter Size/genetics ; Alleles ; }, abstract = {CRISPR/Cas9 technology has been widely utilized to enhance productive performance, increase disease resistance and generate medical models in livestock. The FecB allele in sheep is a mutation in the BMPRIB gene, recognized as the first major gene responsible for the high fecundity trait in sheep, leading to an increased ovulation rate in ewe. In this study, we employed CRISPR/Cas9-mediated homologous-directed repair (HDR) to introduce a defined point mutation (c.746 A > G) using single-stranded oligonucleotides (ssODN) and the ligase IV inhibitor (SCR7) into the BMPRIB gene of fine wool sheep. A total of nine gene-edited sheep were produced, six of which carried the targeted point mutation, with a precise base substitution efficiency (A > G) of 31.6%. Based on the six targeted founders (F0), we expanded the BMPRIB-targeted population, which included F1 heterozygous (B+) and F2 homozygous(BB) or heterozygous offspring. The average litter size of F1 ewes carrying the B + allele reached 170%, comparable to that of heterozygous native Australian Booroola sheep. Gene-edited ewes with B + and BB genotype produced 0.62 and 0.42 more lambs, respectively, compared to wide-type ewes (p < 0.01). Our results also indicated that the parity signification, our data demonstrate that highly efficient introduction of the intended base mutation into the sheep genome can be achieved by combining the CRISPR/Cas9 system with ssODN and SCR7. The offspring of BMPR/B edited sheep with the defined mutation exhibited high fecundity performance. Compared to conventional sheep breeding strategies, genetic improvement through gene editing offered significant advantages without compromising the fine wool traits of Merino sheep, which are often affected by routine cross-breeding methods.}, }
@article {pmid39833307, year = {2025}, author = {Di Cristina, G and Dirksen, E and Altenhein, B and Büschges, A and Korsching, SI}, title = {Pioneering genome editing in parthenogenetic stick insects: CRISPR/Cas9-mediated gene knockout in Medauroidea extradentata.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2584}, pmid = {39833307}, issn = {2045-2322}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Parthenogenesis/genetics ; *Insecta/genetics ; Phenotype ; Female ; Pigmentation/genetics ; }, abstract = {The parthenogenetic life cycle of the stick insect Medauroidea extradentata offers unique advantages for the generation of genome-edited strains, as an isogenic and stable mutant line can in principle be achieved already in the first generation (G0). However, genetic tools for the manipulation of their genes had not been developed until now. Here, we successfully implement CRISPR/Cas9 as a technique to modify the genome of the stick insect M. extradentata. As proof-of-concept we targeted two genes involved in the ommochrome pathway of eye pigmentation (cinnabar and white, second and first exon, respectively), to generate knockout (KO) mutants. Microinjections were performed within 24 h after oviposition, to focus on the mononuclear (and haploid) stage of development. The KOs generated resulted in distinct eye and cuticle colour phenotypes for cinnabar and white. Homozygous cinnabar mutants showed pale pigmentation of eyes and cuticle. They develop into adults capable of producing viable eggs. Homozygous white KO resulted in a completely unpigmented phenotype in developing embryos that were unable to hatch. In conclusion, we show that CRISPR/Cas9 can be successfully applied to the genome of M. extradentata by creating phenotypically different and viable insects. This powerful gene editing technique can now be employed to create stable genetically modified lines using a parthenogenetic non-model organism.}, }
@article {pmid39833279, year = {2025}, author = {Bi, C and Yuan, B and Zhang, Y and Wang, M and Tian, Y and Li, M}, title = {Prevalent integration of genomic repetitive and regulatory elements and donor sequences at CRISPR-Cas9-induced breaks.}, journal = {Communications biology}, volume = {8}, number = {1}, pages = {94}, pmid = {39833279}, issn = {2399-3642}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Regulatory Sequences, Nucleic Acid ; }, abstract = {CRISPR-Cas9 genome editing has been extensively applied in both academia and clinical settings, but its genotoxic risks, including large insertions (LgIns), remain poorly studied due to methodological limitations. This study presents the first detailed report of unintended LgIns consistently induced by different Cas9 editing regimes using various types of donors across multiple gene loci. Among these insertions, retrotransposable elements (REs) and host genomic coding and regulatory sequences are prevalent. RE frequencies and 3D genome organization analysis suggest LgIns originate from randomly acquired genomic fragments by DNA repair mechanisms. Additionally, significant unintended full-length and concatemeric double-stranded DNA (dsDNA) donor integrations occur when donor DNA is present. We further demonstrate that phosphorylated dsDNA donors consistently reduce large insertions and deletions by almost two-fold without compromising homology-directed repair (HDR) efficiency. Taken together, our study addresses a ubiquitous and overlooked risk of unintended LgIns in Cas9 editing, contributing valuable insights for the safe use of Cas9 editing tools.}, }
@article {pmid39833194, year = {2025}, author = {Kim, DG and Gu, B and Cha, Y and Ha, J and Lee, Y and Kim, G and Cho, BK and Oh, MK}, title = {Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {874}, pmid = {39833194}, issn = {2041-1723}, support = {RS-2024-00509338//National Research Foundation of Korea (NRF)/ ; RS-2024-00357320//National Research Foundation of Korea (NRF)/ ; RS-2021-NR056596//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Multigene Family ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Secondary Metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.}, }
@article {pmid39833175, year = {2025}, author = {Goold, HD and Kroukamp, H and Erpf, PE and Zhao, Y and Kelso, P and Calame, J and Timmins, JJB and Wightman, ELI and Peng, K and Carpenter, AC and Llorente, B and Hawthorne, C and Clay, S and van Wyk, N and Daniel, EL and Harrison, F and Meier, F and Willows, RD and Cai, Y and Walker, RSK and Xu, X and Espinosa, MI and Stracquadanio, G and Bader, JS and Mitchell, LA and Boeke, JD and Williams, TC and Paulsen, IT and Pretorius, IS}, title = {Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {841}, pmid = {39833175}, issn = {2041-1723}, support = {DP200100717//Department of Education and Training | Australian Research Council (ARC)/ ; CE200100029//Department of Education and Training | Australian Research Council (ARC)/ ; }, mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Genome, Fungal ; Chromosomes, Artificial, Yeast/genetics ; Chromosomes, Fungal/genetics ; Synthetic Biology/methods ; CRISPR-Cas Systems ; Open Reading Frames/genetics ; RNA, Transfer/genetics/metabolism ; }, abstract = {The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.}, }
@article {pmid39832868, year = {2025}, author = {Alarcón-Iniesta, H and de Arana, G and López-Valls, M and Pardo, D and Escalona-Noguero, C and Rodríguez, C and Castellanos, M and Cobelo, S and Martínez-Ramírez, I and Camarero, J and Heras, SL and de Vicente, J and Valera, A and Smith, W and Bernardo-Gavito, R and Cantón, R and Galán, JC and Granados, D and Miranda, R and Guerrero, H and Sot, B}, title = {CRISPR-associated Plasmonic Colorimeter Method (Ca-PCM): A real-time RGB detection system for gold nanoparticles-based nucleic acid biosensors.}, journal = {Analytica chimica acta}, volume = {1338}, number = {}, pages = {343601}, doi = {10.1016/j.aca.2024.343601}, pmid = {39832868}, issn = {1873-4324}, mesh = {*Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Colorimetry ; *Biosensing Techniques/methods ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {BACKGROUND: The detection of genetic sequences represents the gold standard procedure for species discrimination, genetic characterisation of tumours, and identification of pathogens. The development of new molecular detection methods, accessible and cost effective, is of great relevance. Biosensors based on plasmonic nanoparticles, such as gold nanoparticles (AuNPs), provide a powerful and versatile platform for highly sensitive, economic, user-friendly and label-free sensing. However, the readout techniques typically employed with such sensors lack temporal and kinetic information, which hampers the ability to perform quantitative detection.
RESULTS: In this study, a novel methodology designated the 'CRISPR-associated Plasmonic Colorimeter Method' (Ca-PCM), has been developed. This method combines RNA target recognition by CRISPR LwaCas13a, AuNPs' aggregation, and real-time colorimetric Red-Green-Blue (RGB) analysis. The system registers the AuNP's plasmonic signatures in real-time using a RGB colour sensor with 3-channel silicon photodiodes having blue, green and red sensitivities. The acquired signals are automatically analysed by an algorithm designed to distinguish between positive and negative samples and to correlate the temporal spectral patterns of aggregation with dose-dependent molecular detection of the RNA target. In addition, the combination of Ca-PCM with a previous isothermal amplification allows the target efficient detection in real clinical applications.
SIGNIFICANCE: We have shown that the combination of RGB analysis and continuous temporal measurements is a novel and promising method to characterise the behaviour of gold nanoparticle-based biosensors and to achieve dose-dependent target detection. In addition, the simplicity and cost-effectiveness of this new approach expand the possibilities of other plasmonic-based biosensors and their applicability in low-resources clinical environments.}, }
@article {pmid39832863, year = {2025}, author = {Zhang, T and Wang, X and Jiang, Y and Zhang, Y and Zhao, S and Hu, J and Hu, F and Peng, N}, title = {A miniaturized RPA-CRISPR/Cas12a-based nucleic acid diagnostic platform for rapid and simple self-testing of SARS-CoV-2.}, journal = {Analytica chimica acta}, volume = {1338}, number = {}, pages = {343593}, doi = {10.1016/j.aca.2024.343593}, pmid = {39832863}, issn = {1873-4324}, mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *COVID-19/diagnosis/virology ; CRISPR-Cas Systems/genetics ; Self-Testing ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; RNA, Viral/analysis/genetics ; Limit of Detection ; Molecular Diagnostic Techniques/methods/instrumentation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) RESULTS: In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 min with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) SIGNIFICANCE: This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52).}, }
@article {pmid39832801, year = {2025}, author = {Debnath, A and Sengupta, A and Rudrapal, S and Kumar, A and Rani, M}, title = {In-silico study of molecular adaptations in halophilic Cas9.}, journal = {Letters in applied microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1093/lambio/ovaf006}, pmid = {39832801}, issn = {1472-765X}, abstract = {This study explores the structural adaptations of the CRISPR-Cas9 system in halophilic bacteria, focusing on Cas9 protein of halophilic bacterium Salicibibacter cibi. Protein sequences were analysed using different tools such as ExPASy ProtParam for different physicochemical properties, PONDR web server for disordered regions, and InterPro server and WebLogo for domains. Protein structures were generated using the AlphaFold database, and the quality of the modelled structure was checked through PROCHECK. The protein surface's amino acids and electrostatic potential were visualized using PyMOL, APBS server and UCSF chimera. Comparative analysis revealed that halophilic Cas9 proteins possess a higher abundance of acidic residues, resulting in enhanced stability and hydration in saline conditions; halophilic Cas9 proteins also shows higher intrinsically disordered regions. Electrostatic potential maps confirmed that S. cibi Cas9 proteins maintain a highly negative surface charge, crucial for adaptation to salt-rich environments. These findings provide insights into the molecular mechanisms driving the structural and functional adaptations of Cas9 in salty environment, highlighting its potential applications in genome editing-based biotechnological approaches in extreme conditions.}, }
@article {pmid39832424, year = {2025}, author = {Choudhary, R and Ahmad, F and Kaya, C and Upadhyay, SK and Muneer, S and Kumar, V and Meena, M and Liu, H and Upadhyaya, H and Seth, CS}, title = {Decrypting proteomics, transcriptomics, genomics, and integrated omics for augmenting the abiotic, biotic, and climate change stress resilience in plants.}, journal = {Journal of plant physiology}, volume = {305}, number = {}, pages = {154430}, doi = {10.1016/j.jplph.2025.154430}, pmid = {39832424}, issn = {1618-1328}, abstract = {As our planet faces increasing environmental challenges, such as biotic pressures, abiotic stressors, and climate change, it is crucial to understand the complex mechanisms that underlie stress responses in crop plants. Over past few years, the integration of techniques of proteomics, transcriptomics, and genomics like LC-MS, IT-MS, MALDI-MS, DIGE, ESTs, SAGE, WGS, GWAS, GBS, 2D-PAGE, CRISPR-Cas, cDNA-AFLP, HLS, HRPF, MPSS, CAGE, MAS, IEF, MudPIT, SRM/MRM, SWATH-MS, ESI have significantly enhanced our ability to comprehend the molecular pathways and regulatory networks, involved in balancing the ecosystem/ecology stress adaptation. This review offers thorough synopsis of the current research on utilizing these multi-omics methods (including metabolomics, ionomics) for battling abiotic (salinity, temperature (chilling/freezing/cold/heat), flood (hypoxia), drought, heavy metals/loids), biotic (pathogens like fungi, bacteria, virus, pests, and insects (aphids, caterpillars, moths, mites, nematodes) and climate change stress (ozone, ultraviolet radiation, green house gases, carbon dioxide). These strategies can expedite crop improvement, and act as powerful tools with high throughput and instant database generation rates. They also provide a platform for interpreting intricate, systematic signalling pathways and knowing how different environmental stimuli cause phenotypic responses at cellular and molecular level by changing the expression of stress-responsive genes like RAB18, KIN1, RD29B, OsCIPK03, OsSTL, SIAGL, bZIP, SnRK, ABF. This review discusses various case studies that exemplify the successful implementation of these omics tools to enhance stress tolerance in plants. Finally, it highlights challenges and future prospects of utilizing these approaches in combating stress, emphasizing the need for interdisciplinary collaborations and bio-technological advancements for sustainable agriculture and food security.}, }
@article {pmid39832407, year = {2025}, author = {Mao, Z and Chen, R and Huang, L and Ren, S and Liu, B and Gao, Z}, title = {CRISPR analysis based on Pt@MOF dual-modal signal for multichannel fluorescence and visual detection of norovirus.}, journal = {Biosensors & bioelectronics}, volume = {273}, number = {}, pages = {117153}, doi = {10.1016/j.bios.2025.117153}, pmid = {39832407}, issn = {1873-4235}, abstract = {Norovirus is a globally prevalent pathogen that causes acute viral gastroenteritis across all age groups, characterized by its high infectivity and low infectious dose. Consequently, the development of rapid, sensitive, and accurate detection technologies for norovirus presents a significant challenge. In this study, we demonstrate a combination of CRISPR-Cas-based reactions with Pt@MOF-linked immunoassay-like assays. This methodology enables both qualitative analysis and colorimetric readouts of Cas12a-mediated DNA/RNA detection at room temperature, as well as the generation of fluorescent signal readout through base deprotonation-induced Pt@MOF cleavage of a fluorogenic substrate. Furthermore, the integration of RPA amplification with noncanonical PAM-designed CRISPR significantly enhances the sensitivity and flexibility of detection, facilitating the extension of this strategy to other targets. Ultimately, the strategy was validated in spiked food samples with a 100% accuracy rate, consistent with RT-qPCR results. Collectively, this work showcases a viable approach for a dual-functional Pt@MOF-based CRISPR biosensing platform for bioanalysis and a flexible, universal strategy based on noncanonical PAM-designed gRNAs.}, }
@article {pmid39764982, year = {2025}, author = {Yue, Y and Liu, M and Ma, M and Xu, Z and Zhang, H and Wang, Q and Liu, R}, title = {CRISPR/Cas14a integrated with DNA walker based on magnetic self-assembly for human papillomavirus type 16 oncoprotein E7 ultrasensitive detection.}, journal = {Biosensors & bioelectronics}, volume = {272}, number = {}, pages = {117135}, doi = {10.1016/j.bios.2025.117135}, pmid = {39764982}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Papillomavirus E7 Proteins/genetics ; Animals ; Humans ; Mice ; *Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Limit of Detection ; Papillomavirus Infections/virology/diagnosis ; Electrochemical Techniques/methods ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; }, abstract = {To enhance the biomarker diagnostics sensitivity and selectivity of human papillomavirus type 16 oncoprotein E7 (HPV16 E7) in serum, a label/enzyme-free electrochemical detection platform was developed. This platform featured a type of "Super-turn-off" nanobiosensor monitored through differential pulse voltammetry (DPV). It integrated the magnetic self-assembly property of the α-Fe2O3/Fe3O4@Au/Sub/BSA signal transport nano-medium with the high specificity of CRISPR/Cas14a and the amplification capability of the bipedal walker (DNA walker composed of two ssDNA strands), resulting in the enhanced specificity and anti-interference performance while remaining stable at 4 °C for over 30 days. The results demonstrated that the combination of walker and CRISPR yielded superior sensitivity and analytical capability compared with using either technology alone, achieving a detection limit of 67.17 fg mL[-1], a quantification limit of 0.22 pg mL[-1], and serum sample recovery rates of 98.46%-115.78%. Additionally, this platform was applied to detect serum and tissue samples from mouse models at various stages of cervical cancer, significantly improving the accuracy and effectiveness of early diagnosis and prognostic evaluation. This novel approach held promise as an efficient tool for point-of-care clinical detection of HPV16 E7, potentially reducing cervical cancer mortality.}, }
@article {pmid39740588, year = {2025}, author = {Liu, H and Lv, MM and Li, X and Su, M and Nie, YG and Ying, ZM}, title = {Ligation-recognition triggered RPA-Cas12a cis-cleavage fluorogenic RNA aptamer for one-pot and label-free detection of MicroRNA in breast cancer.}, journal = {Biosensors & bioelectronics}, volume = {272}, number = {}, pages = {117106}, doi = {10.1016/j.bios.2024.117106}, pmid = {39740588}, issn = {1873-4235}, mesh = {*Breast Neoplasms/genetics/diagnosis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/blood/genetics/analysis ; Female ; Nucleic Acid Amplification Techniques/methods ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Limit of Detection ; Bacterial Proteins/chemistry/genetics ; CRISPR-Associated Proteins/chemistry/genetics ; Endodeoxyribonucleases/chemistry ; }, abstract = {"One-pot" assays which combine amplification with CRISPR/Cas12a system are in constant attracted for biosensors development. Herein, we present a one-pot isothermal assay that Ligation-recognition triggered Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a cis-cleavage (LRPA-CRISPR) fluorescent biosensor for sensitive, specific, and label-free miRNA detection. Firstly, we reveal the programmed double-stranded DNA amplicons, which utilized the ligation-recognition and polymerization to form and amplified by the RPA system. Meanwhile, we enabled exponential ligation-recognition triggered recombinase polymerase amplification of miRNA-21 sequences and exploited the cis-cleavage mechanism of Cas12a with transcription to generate functional Mango RNA for signal output. This assay can be completed within 40 min and can allow a limit of detection of 3.43 aM for miRNA-21 detection, owing to the RPA with transcription amplification and enables to product the functional Mango RNA aptamer by in vitro transcription that binds to the TO1-Biotin fluorogenic dye. Moreover, our method exhibits the advantages of self-supply crRNA, label-free, excellent specificity, and universal detection platform via the design of one-pot detection in serum and cell samples, showing tremendous potential in biomarkers diagnostics of breast cancer.}, }
@article {pmid39622311, year = {2025}, author = {Kim, M and Hutchins, EJ}, title = {CRISPR-Cas13d as a molecular tool to achieve targeted gene expression knockdown in chick embryos.}, journal = {Developmental biology}, volume = {519}, number = {}, pages = {5-12}, doi = {10.1016/j.ydbio.2024.11.013}, pmid = {39622311}, issn = {1095-564X}, support = {R00 DE028592/DE/NIDCR NIH HHS/United States ; R35 GM150763/GM/NIGMS NIH HHS/United States ; T32 DE007306/DE/NIDCR NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Chick Embryo ; *Gene Knockdown Techniques/methods ; *PAX7 Transcription Factor/genetics/metabolism ; Neural Crest/metabolism/embryology ; Gene Expression Regulation, Developmental/genetics ; Morpholinos/genetics ; }, abstract = {The chick embryo is a classical model system commonly used in developmental biology due to its amenability to gene perturbation experiments. Pairing this powerful model organism with cutting-edge technology can significantly expand the range of experiments that can be performed. Recently, the CRISPR-Cas13d system has been successfully adapted for use in zebrafish, medaka, killifish, and mouse embryos to achieve targeted gene expression knockdown. Despite its success in other animal models, no prior study has explored the potential of CRISPR-Cas13d in the chick. Here, we present an adaptation of the CRISPR-Cas13d system to achieve targeted gene expression knockdown in the chick embryo. As proof-of-principle, we demonstrate the knockdown of PAX7, an early neural crest marker. Application of this adapted CRISPR-Cas13d technique resulted in effective knockdown of PAX7 expression and function, comparable to knockdown achieved by translation-blocking morpholino. CRISPR-Cas13d complements preexisting knockdown tools such as CRISPR-Cas9 and morpholinos, thereby expanding the experimental potential and versatility of the chick model system.}, }
@article {pmid39313516, year = {2025}, author = {Liang, YL and Hu, YX and Li, FF and You, HM and Chen, J and Liang, C and Guo, ZF and Jing, Q}, title = {Adaptor protein Src-homology 2 domain containing E (SH2E) deficiency induces heart defect in zebrafish.}, journal = {Acta pharmacologica Sinica}, volume = {46}, number = {2}, pages = {404-415}, pmid = {39313516}, issn = {1745-7254}, mesh = {Animals ; *Zebrafish ; *Zebrafish Proteins/genetics/metabolism/deficiency ; Heart Defects, Congenital/genetics/metabolism ; NF-kappa B/metabolism ; Adaptor Proteins, Signal Transducing/genetics/metabolism ; Signal Transduction ; CRISPR-Cas Systems ; }, abstract = {Adaptor proteins play crucial roles in signal transduction across diverse signaling pathways. Src-homology 2 domain-containing E (SH2E) is the adaptor protein highly expressed in vascular endothelial cells and myocardium during zebrafish embryogenesis. In this study we investigated the function and mechanisms of SH2E in cardiogenesis. We first analyzed the spatiotemporal expression of SH2E and then constructed zebrafish lines with SH2E deficiency using the CRISPR-Cas9 system. We showed that homozygous mutants developed progressive pericardial edema (PCE), dilated atrium, abnormal atrioventricular looping and thickened atrioventricular wall from 3 days post fertilization (dpf) until death; inducible overexpression of SH2E was able to partially rescue the PCE phenotype. Using transcriptome sequencing analysis, we demonstrated that the MAPK/ERK and NF-κB signaling pathways might be involved in SH2E-deficiency-caused PCE. This study underscores the pivotal role of SH2E in cardiogenesis, and might help to identify innovative diagnostic techniques and therapeutic strategies for congenital heart disease.}, }
@article {pmid39831708, year = {2025}, author = {Cochrane, RW and Robino, RA and Ferreira, LMR}, title = {Generation of Human Chimeric Antigen Receptor Regulatory T Cells.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {215}, pages = {}, doi = {10.3791/67200}, pmid = {39831708}, issn = {1940-087X}, mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *Receptors, Chimeric Antigen/immunology/genetics ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Dependovirus/genetics/immunology ; }, abstract = {Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4[+] T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs' immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.}, }
@article {pmid39831695, year = {2025}, author = {Yan, C and Meng, H and Pei, Y and Sun, W and Zhang, J}, title = {Breeding by Design for Functional Rice with Genome Editing Technologies.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {215}, pages = {}, doi = {10.3791/67336}, pmid = {39831695}, issn = {1940-087X}, mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; *CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; }, abstract = {The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.}, }
@article {pmid39831307, year = {2025}, author = {Fei, X and Lei, C and Ren, W and Liu, C}, title = {'Splice-at-will' Cas12a crRNA engineering enabled direct quantification of ultrashort RNAs.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkaf002}, pmid = {39831307}, issn = {1362-4962}, support = {22074088//National Natural Science Foundation of China/ ; IRT_15R43//Program for Changjiang Scholars and Innovative Research Team in University/ ; GK202305001//Fundamental Research Funds for the Central Universities/ ; //Shaanxi Normal University/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; RNA/genetics/chemistry/metabolism ; RNA Splicing ; Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics/chemistry ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; }, abstract = {We present a robust 'splice-at-will' CRISPR RNA (crRNA) engineering mechanism that overcomes the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system in directly detecting ultrashort RNAs. In this strategy, an intact Cas12a crRNA can be split from almost any site of the spacer region to obtain a truncated crRNA (tcrRNA) that cannot activate Cas12a even after binding an auxiliary DNA activator. While splicing tcrRNAs with a moiety of ultrashort RNA, the formed combination can work together to activate Cas12a efficiently, enabling 'splice-at-will' crRNA engineering. Importantly, the 'splice-at-will' crRNA exhibits almost the same trans-cleavage activation efficiency as that of a conventional intact crRNA. Therefore, by rationally designing a DNA auxiliary activator with a conserved tcrRNA-complementary sequence and an arbitrary short RNA-of-interest recognition domain, a general sensing system is established that directly utilizes traditional DNA-activated Cas12a to detect ultrashort RNAs. This 'splice-at-will' crRNA engineering strategy could faithfully detect ultrashort RNA sequences as short as 6-8 nt, which cannot be achieved by conventional Cas12a and Cas13a systems. Additionally, through flexible splicing site design, our method can precisely distinguish single-base differences in microRNA and other short RNA sequences. This work has significantly expanded the Cas12a-based diagnostic toolbox and opened new avenues for ultrashort RNA detection.}, }
@article {pmid39829748, year = {2025}, author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Estevam, GO and Kim, N and Hill, E and Ruffolo, JA and Silverstein, R and Gallagher, J and Kleinstiver, B and Meeske, AJ and Cameron, P and Madani, A}, title = {Engineering of CRISPR-Cas PAM recognition using deep learning of vast evolutionary data.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2025.01.06.631536}, pmid = {39829748}, issn = {2692-8205}, abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, significantly limiting the range of targetable sequences in a genome. Machine learning-based protein engineering provides a powerful solution to efficiently generate Cas protein variants tailored to recognize specific PAMs. Here, we present Protein2PAM, an evolution-informed deep learning model trained on a dataset of over 45,000 CRISPR-Cas PAMs. Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across Type I, II, and V CRISPR-Cas systems. Using in silico deep mutational scanning, we demonstrate that the model can identify residues critical for PAM recognition in Cas9 without utilizing structural information. As a proof of concept for protein engineering, we employ Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild-type under in vitro conditions. This work represents the first successful application of machine learning to achieve customization of Cas enzymes for alternate PAM recognition, paving the way for personalized genome editing.}, }
@article {pmid39829431, year = {2025}, author = {Chen, Y and Xue, Y and Jiang, Q and Jin, Y and Chen, W and Hua, M}, title = {Disruption of the FOXM1 Regulatory Region Inhibits Tumor Progression in Ovarian Cancer by CRISPR-Cas9.}, journal = {Drug development research}, volume = {86}, number = {1}, pages = {e70049}, doi = {10.1002/ddr.70049}, pmid = {39829431}, issn = {1098-2299}, support = {//This work was supported by the Nantong Social Science and Technology Plan for People's Livelihood Project (grant No. JCZ2023003) from the Science and Technology Bureau of Nantong, China, and the Research Physician Development Fund (grant No. YJXYY202204-YSC03) from the Affiliated Hospital of Nantong University, China./ ; }, mesh = {Female ; *Forkhead Box Protein M1/genetics ; Humans ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Mice ; *Cell Proliferation/drug effects ; Azepines/pharmacology ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Movement/drug effects ; Triazoles/pharmacology ; Xenograft Model Antitumor Assays ; Disease Progression ; }, abstract = {Ovarian cancer is the seventh most common lethal tumor among women in the world. FOXM1 is a transcription factor implicated in the initiation and progression of ovarian cancer by regulating key oncogenic genes. The role of regulatory regions in regulating the expression of FOXM1 in ovarian cancer is not completely clarified. Treatment with bromodomain and extraterminal (BET) inhibitors JQ-1 and I-BET were explored in ovarian cancer cell lines (OVCAR3, A2780, or SKOV3) to evaluate FOXM1 expression and biological behavior by qPCR, CCK8 assay, colony formation assay, wound-healing, and transwell assays. The regulatory regions (enhancer sequence spanning promoter or exon 1) of FOXM1 were deleted using CRISPR-Cas9 in the OVCAR3 cell line. FOXM1 expression and tumor biological behavior were further assessed in FOXM1 regulatory regions deleted OVCAR3 cell line. The mouse xenograft model was assessed at the indicated time points following subcutaneous injection of enhancer-deleted cells. Treatment with the JQ-1 and I-BET reduced the expression of FOXM1, decreasing cell proliferation, migration, and invasion in a panel of ovarian cancer cell lines including OVCAR3, A2780, and SKOV3 cells. By mining the published ChIP-sequencing data (H3K27Ac) from 12 ovarian cancer cell lines, we identified a potential enhancer and promoter region. Deletion of the spanning enhancer and promoter region of FOXM1 reduced mRNA and protein expression. Similarly, cell proliferation, migration, invasion, and tumorigenesis in both cells and mouse xenograft models were significantly attenuated. Our study demonstrates that JQ-1 and I-BET can regulate the expression of the FOXM1 gene-relating network. These data also indicate that disruption of the span enhancer and promoter region activity of FOXM1 has a vital role in the anti-ovarian cancer effect, hiding a potential opportunity for the evaluation of this non-coding DNA deletion disrupts the FOXM1 transcriptional network in ovarian cancer development.}, }
@article {pmid39828694, year = {2025}, author = {Matthews, MC and van der Linden, J and Robène, I and Rozsasi, S and Coetzee, B and Campa, M and Burger, J and Akwuruoha, UN and Madufor, NJ and Perold, W and Opara, UL and Viljoen, A and Mostert, D}, title = {A combined recombinase polymerase amplification CRISPR/Cas12a assay for detection of Fusarium oxysporum f. sp. cubense tropical race 4.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2436}, pmid = {39828694}, issn = {2045-2322}, support = {138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 138109//National Research Foundation/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; 890856//Horizon 2020 Framework Programme/ ; }, mesh = {*CRISPR-Cas Systems ; *Fusarium/genetics/isolation & purification ; *Musa/microbiology ; Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; Recombinases/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The soilborne pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is currently devastating banana production worldwide. Once introduced, it is not possible to eradicate the pathogen from soils where it can survive for decades. The only management option available then is to replace Foc TR4-susceptible with -resistant varieties. Timely detection of the pathogen, however, is an important strategy to prevent the introduction of Foc TR4 into new areas and prevent its spread from infested sites. In this study, a single-tube detection technique was developed by combining recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology (RPA-Cas12a) for detection of Foc TR4. The RPA-Cas12a assay was conducted isothermally, had a sensitivity of up to 10 fg target DNA and did not cross react with any of the 76 non-target isolates included in the specificity testing. The RPA-Cas12a assay detected Foc TR4 from naturally infected banana samples collected in the field and visualization was possible with the naked eye under LED blue light transillumination. The method can be integrated with inexpensive fluorescent or electronic detection devices to accelerate Foc TR4 in-field detection and, thereby, fast-track disease containment strategies.}, }
@article {pmid39827180, year = {2025}, author = {Van Goethem, MW and Bezuidt, OKI and Pierneef, R and Vikram, S and Hopkins, DW and Aspray, T and Hall, G and Woodborne, S and Hogg, ID and Northen, TR and Kong, W and Daffonchio, D and Cowan, DA and Van de Peer, Y and Delgado-Baquerizo, M and Makhalanyane, TP}, title = {Novel adaptive immune systems in pristine Antarctic soils.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {2368}, pmid = {39827180}, issn = {2045-2322}, support = {118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 118981//National Research Foundation/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; 110717//South African National Antarctic Programme/ ; }, mesh = {Antarctic Regions ; *Soil Microbiology ; *Phylogeny ; *CRISPR-Cas Systems ; Microbiota/genetics ; Metagenomics/methods ; Adaptive Immunity/genetics ; Genome, Bacterial ; Bacteria/genetics ; Prophages/genetics ; Metagenome ; }, abstract = {Antarctic environments are dominated by microorganisms, which are vulnerable to viral infection. Although several studies have investigated the phylogenetic repertoire of bacteria and viruses in these poly-extreme environments with freezing temperatures, high ultra violet irradiation levels, low moisture availability and hyper-oligotrophy, the evolutionary mechanisms governing microbial immunity remain poorly understood. Using genome-resolved metagenomics, we test the hypothesis that Antarctic poly-extreme high-latitude microbiomes harbour diverse adaptive immune systems. Our analysis reveals the prevalence of prophages in bacterial genomes (Bacteroidota and Verrucomicrobiota), suggesting the significance of lysogenic infection strategies in Antarctic soils. Furthermore, we demonstrate the presence of diverse CRISPR-Cas arrays, including Class 1 arrays (Types I-B, I-C, and I-E), alongside systems exhibiting novel gene architecture among their effector cas genes. Notably, a Class 2 system featuring type V variants lacks CRISPR arrays, encodes Cas1 and Cas2 adaptation module genes. Phylogenetic analysis of Cas12 effector proteins hints at divergent evolutionary histories compared to classified type V effectors and indicates that TnpB is likely the ancestor of Cas12 nucleases. Our findings suggest substantial novelty in Antarctic cas sequences, likely driven by strong selective pressures. These results underscore the role of viral infection as a key evolutionary driver shaping polar microbiomes.}, }
@article {pmid39826761, year = {2025}, author = {Zhou, Z and Liu, S and Saleem, M and Liu, F and Hu, R and Su, H and Dong, D and Luo, Z and Wu, Y and Zhang, Y and He, Z and Wang, C}, title = {Unraveling phase-dependent variations of viral community, virus-host linkage, and functional potential during manure composting process.}, journal = {Bioresource technology}, volume = {}, number = {}, pages = {132081}, doi = {10.1016/j.biortech.2025.132081}, pmid = {39826761}, issn = {1873-2976}, abstract = {The temporal dynamics of bacterial and fungal communities significantly impact the manure composting process, yet viral communities are often underexplored. Bulk metagenomes, viromes, metatranscriptomes, and metabolomes were integrated to investigate dynamics of double-stranded DNA (dsDNA) virus and virus-host interactions throughout a 63-day composting process. A total of 473 viral operational taxonomic units (vOTUs), predominantly Caudoviricetes, showed distinct phase-dependent differentiation. In phase I (initial-mesophilic), viruses targeted Gammaproteobacteria and Firmicutes, utilizing restriction-modification (RM) systems. In phase II (thermophilic-maturing), viruses infected Alphaproteobacteria, Chloroflexi, and Planctomycetes, employing CRISPR-Cas systems. Lysogenic and lytic viruses exerting differential effects on bacterial pathogens across phases. Additionally, six types of auxiliary metabolic genes (AMGs) related to galactose and cysteine metabolisms were identified. The homologous lineages of AMGs with bacterial genes, along with the significant temporal correlation observed between virus-host-metabolite interactions, underscore the critical yet often overlooked role of viral communities in modulating microbial metabolisms and pathogenesis within composting ecosystems.}, }
@article {pmid39826547, year = {2025}, author = {Hong, SH and An, SY and Park, C and Kim, Y and Kim, EH and Kim, NK and Suh, JY}, title = {Structural variants of AcrIIC5 inhibit Cas9 via divergent binding interfaces.}, journal = {Structure (London, England : 1993)}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.str.2024.12.014}, pmid = {39826547}, issn = {1878-4186}, abstract = {CRISPR-Cas is a bacterial defense system that employs RNA-guided endonucleases to destroy invading foreign nucleic acids. Bacteriophages produce anti-CRISPR (Acr) proteins to evade CRISPR-Cas defense during the infection. AcrIIC5, a type II-C Cas9 inhibitor, exhibits unusual variations in the local backbone fold between its orthologs. Here we investigated how the folding variations affect the inhibition of target Cas9 using AcrIIC5 orthologs. Structural comparison of free AcrIIC5Smu and AcrIIC5Nch confirmed that the folding variation correlated with characteristic indels in the helical region. Mutagenesis and biochemical assays combined with AlphaFold2 predictions identified key residues of AcrIIC5 orthologs important for Cas9 inhibition. Remarkably, AcrIIC5 orthologs employed divergent binding interfaces via folding variations to inhibit the Cas9 targets. Our study suggests that Acr proteins have evolved structural variants to diversify key interfaces for target Cas9, which could be beneficial for the adaptation of phages to evasive mutations on the Cas9 surface.}, }
@article {pmid39700063, year = {2025}, author = {Munusamy, S and Zheng, H and Jahani, R and Zhou, S and Chen, J and Kong, J and Guan, X}, title = {DNA-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay for Protein Detection in Complicated Matrices.}, journal = {ACS applied bio materials}, volume = {8}, number = {1}, pages = {754-762}, doi = {10.1021/acsabm.4c01600}, pmid = {39700063}, issn = {2576-6422}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry ; Materials Testing ; Biocompatible Materials/chemistry ; Particle Size ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases/metabolism/genetics ; Proteins/analysis/metabolism ; Bacterial Proteins ; }, abstract = {Proteins are important biological macromolecules that perform a wide variety of functions in the cell and human body, and can serve as important biomarkers for early diagnosis and prognosis of human diseases as well as monitoring the effectiveness of disease treatment. Hence, sensitive and accurate detection of proteins in human biospecimens is imperative. However, at present, there is no ideal method available for the detection of proteins in clinical samples, many of which are present at ultralow (less than 1 pM) concentrations and in complicated matrices. Herein, we report an ultrasensitive and selective DNA-assisted CRISPR-Cas12a enhanced fluorescent assay (DACEA) for protein detection with detection limits reaching as low as attomolar concentrations. The high assay sensitivity was accomplished through the combined DNA barcode amplification (by using dual-functionalized AuNPs) and CRISPR analysis, while the high selectivity and high resistance to the matrix effects of our method were accomplished via the formation of protein-antibody sandwich structure and the specific recognition of Cas12a (under the guidance of crRNA) toward the designed target ssDNA. Given its ability to accurately and sensitively detect trace amounts of proteins in complicated matrices, the DACEA protein assay platform pioneered in this work has a potential application in routine protein biomarker testing.}, }
@article {pmid39825389, year = {2025}, author = {Abraham, IC and Aboje, JE and Ukoaka, BM and Tom-Ayegunle, K and Amjad, M and Abdulkader, A and Agbo, CE and Akinruli, OA and Akisanmi, TR and Oyetola, EO and Olatunji, G and Kokori, E and Aderinto, N}, title = {Integrating malaria vaccine and CRISPR/Cas9 gene drive: a comprehensive strategy for accelerated malaria eradication.}, journal = {Malaria journal}, volume = {24}, number = {1}, pages = {17}, pmid = {39825389}, issn = {1475-2875}, mesh = {*Malaria Vaccines/immunology/genetics/administration & dosage ; *CRISPR-Cas Systems ; *Malaria/prevention & control ; *Gene Drive Technology/methods ; Humans ; *Disease Eradication/methods ; Animals ; Anopheles/genetics/parasitology ; Mosquito Vectors/genetics ; Mosquito Control/methods ; }, abstract = {Malaria remains a significant public health challenge, particularly in low- and middle-income countries, despite ongoing efforts to eradicate the disease. Recent advancements, including the rollout of malaria vaccines, such as RTS,S/AS01 and R21/Matrix-M™, offer new avenues for prevention. However, the rise of resistance to anti-malarial medications necessitates innovative strategies. This review explores the potential integration of CRISPR/Cas9 gene drive technology with malaria vaccination efforts to enhance vector control and reduce transmission. By employing gene drive mechanisms for population suppression and replacement of malaria-transmitting Anopheles mosquitoes, combined with the immunogenic properties of vaccines, a synergistic approach can be established. This paper discussed the need for integrated strategies to address the biological complexities of malaria and socio-economic factors influencing its prevalence. Challenges such as regulatory hurdles, community acceptance, ecological impacts, and sustainable funding are examined, alongside strategies for implementation within existing malaria control programmes. This integrated approach could significantly contribute to achieving the World Health Organization's targets for malaria reduction by 2030, ultimately enhancing public health outcomes and supporting broader socio-economic development.}, }
@article {pmid39824586, year = {2025}, author = {Arivarasan, VK and Diwakar, D and Kamarudheen, N and Loganathan, K}, title = {Current approaches in CRISPR-Cas systems for diabetes.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {95-125}, doi = {10.1016/bs.pmbts.2024.08.002}, pmid = {39824586}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Diabetes Mellitus/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; }, abstract = {In the face of advancements in health care and a shift towards healthy lifestyle, diabetes mellitus (DM) still presents as a global health challenge. This chapter explores recent advancements in the areas of genetic and molecular underpinnings of DM, addressing the revolutionary potential of CRISPR-based genome editing technologies. We delve into the multifaceted relationship between genes and molecular pathways contributing to both type1 and type 2 diabetes. We highlight the importance of how improved genetic screening and the identification of susceptibility genes are aiding in early diagnosis and risk stratification. The spotlight then shifts to CRISPR-Cas9, a robust genome editing tool capable of various applications including correcting mutations in type 1 diabetes, enhancing insulin production in T2D, modulating genes associated with metabolism of glucose and insulin sensitivity. Delivery methods for CRISPR to targeted tissues and cells are explored, including viral and non-viral vectors, alongside the exciting possibilities offered by nanocarriers. We conclude by discussing the challenges and ethical considerations surrounding CRISPR-based therapies for DM. These include potential off-target effects, ensuring long-term efficacy and safety, and navigating the ethical implications of human genome modification. This chapter offers a comprehensive perspective on how genetic and molecular insights, coupled with the transformative power of CRISPR, are paving the way for potential cures and novel therapeutic approaches for DM.}, }
@article {pmid39824585, year = {2025}, author = {Munshi, ID and Acharya, M and Mukherjee, S and Mani, I}, title = {Recent development in CRISPR-Cas systems for cardiac disease.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {47-93}, doi = {10.1016/bs.pmbts.2024.08.004}, pmid = {39824585}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Heart Diseases/genetics/therapy ; *Gene Editing ; }, abstract = {The CRISPR-Cas system has emerged as a revolutionary tool in genetic research, enabling highly precise gene editing and significantly advancing the field of cardiovascular science. This chapter provides a comprehensive overview of the latest developments in utilizing CRISPR-Cas technologies to investigate and treat heart diseases. It delves into the application of CRISPR-Cas9 for creating accurate models of complex cardiac conditions, such as hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and various arrhythmias, which are essential for understanding disease mechanisms and testing potential therapies. The therapeutic potential of gene editing is also explored, with a focus on genes like PCSK9 and ANGPTL3 that play critical roles in lipid metabolism and cardiovascular health, offering promising avenues for new treatments. Furthermore, the expanding applications of CRISPR in heart tissue regeneration are examined, which could revolutionize the repair of damaged heart tissue. Cutting-edge techniques such as base editing and prime editing are discussed, highlighting their potential to further refine genetic interventions. The discussion concludes by addressing the challenges associated with delivering CRISPR components efficiently and safely, while also exploring recent innovations that may overcome these hurdles, providing insights into the future directions of CRISPR technology in cardiovascular medicine.}, }
@article {pmid39824584, year = {2025}, author = {Khoshandam, M and Soltaninejad, H and Bhia, I and Goudarzi, MTH and Hosseinkhani, S}, title = {CRISPR challenges in clinical developments.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {263-279}, doi = {10.1016/bs.pmbts.2024.08.001}, pmid = {39824584}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Genetic Therapy/methods ; Neoplasms/genetics/therapy ; }, abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is a novel genome editing technology with potential applications in treating diseases. Currently, its use in humans is restricted to clinical trials, although its growth rate is significant, and some have received initial FDA approval. It is crucial to examine and address the challenges for this technology to be implemented in clinical settings. This review aims to identify and explore new research ideas to increase of CRISPR's efficiency in treating genetic diseases and cancer, as well as its future prospects. Given that a substantial amount of previous research has focused on CRISPR-Cas delivery strategies and materials, this overview introduces specific conditions and strategies. It also discusses some of the challenges and opportunities in this field, offering a unique perspective.}, }
@article {pmid39824583, year = {2025}, author = {Mishra, S and Banerjee, S and Tiwari, BS and Tiwari, AK}, title = {Recent progress in CRISPR-Cas-system for neurological disorders.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {231-261}, doi = {10.1016/bs.pmbts.2024.07.017}, pmid = {39824583}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nervous System Diseases/genetics/therapy ; Animals ; Gene Editing ; Genetic Therapy/methods ; }, abstract = {Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.}, }
@article {pmid39824582, year = {2025}, author = {Gowda, DAA and Birappa, G and Rajkumar, S and Ajaykumar, CB and Srikanth, B and Kim, SL and Singh, V and Jayachandran, A and Lee, J and Ramakrishna, S}, title = {Recent progress in CRISPR/Cas9 system for eye disorders.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {21-46}, doi = {10.1016/bs.pmbts.2024.07.018}, pmid = {39824582}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Eye Diseases/genetics/therapy ; Gene Editing ; }, abstract = {Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.}, }
@article {pmid39824581, year = {2025}, author = {Singh, S and Raj, D and Mathur, A and Mani, N and Kumar, D}, title = {Current approaches in CRISPR-Cas systems for hereditary diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {205-229}, doi = {10.1016/bs.pmbts.2024.07.015}, pmid = {39824581}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genetic Diseases, Inborn/therapy/genetics ; *Gene Editing/methods ; Animals ; }, abstract = {CRISPR-Cas technologies have drastically revolutionized genetic engineering and also dramatically changed the potential for treating inherited disorders. The potential to correct genetic mutations responsible for numerous hereditary disorders from single-gene disorders to complex polygenic diseases through precise DNA editing is feasible. The tactic now employed in CRISPR-Cas systems for treating inherited disorders is the usage of particular guide RNAs to target and edit disease-causing mutations in the patient's genome. Several methods such as CRISPR-Cas9, CRISPR-Cas12, and CRISPR-Cas13 are being thoroughly researched and optimized to increase effectiveness, accuracy, and safety in gene editing. Additionally, it is predicted that CRISPR-based therapies will be able to treat complex genetic illnesses such as cancer predisposition syndromes, neurological disorders, and cardiovascular conditions in addition to single-gene disorders. The available editing tools and creation of base editing technology facilitate the simultaneous correction of many mutations or accurate nucleotide changes leading to further advances in the development of multiplex editing tools and base editing technology fiction. When combined with other paradigms such as gene therapy using stem cell treatment, CRISPR-Cas promises improved efficacy. Patient treatment and lowering side effects significantly in individual genetic profiles will guide CRISPR-based treatments. These procedures will undoubtedly lead to therapies that are both efficient and curative of a wide range of genetic diseases, ushering in a new era of precision medicine. This chapter discusses about CRISPR Cas9 mechanism and its significance in the treatment of Hereditary disorders.}, }
@article {pmid39824580, year = {2025}, author = {Israr, J and Kumar, A}, title = {Current progress in CRISPR-Cas systems for rare diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {163-203}, doi = {10.1016/bs.pmbts.2024.07.019}, pmid = {39824580}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Rare Diseases/genetics/therapy ; Animals ; *Gene Editing/methods ; Genetic Therapy/methods ; }, abstract = {The groundbreaking CRISPR-Cas gene editing method permits exact genetic code alteration. The "CRISPR" DNA protects bacteria from viruses. CRISPR-Cas utilizes a guide RNA to steer the Cas enzyme to the genome's gene editing target. After attaching to a sequence, Cas enzymes cleave DNA to insert, delete, or modify genes. The influence of CRISPR-Cas technology on molecular biology and genetics is profound. It allows for gene function research, animal disease models, and patient genetic therapy. Gene editing has transformed biotechnology, agriculture, and customized medicine. CRISPR-Cas could revolutionize genetics and medicine. CRISPR-Cas may accurately correct genetic flaws that underlie rare diseases, improving their therapy. Gene mutations make CRISPR-Cas gene editing a viable cure for uncommon diseases. We can use CRISPR-Cas to correct genetic abnormalities at the molecular level. This strategy offers hope for remedies and disease understanding. CRISPR-Cas genome editing may enable more targeted and effective treatments for rare medical illnesses with few therapy options. By developing base- and prime-editing CRISPR technology, CRISPR-Cas allows for accurate and efficient genome editing and advanced DNA modification. This advanced method provides precise DNA alterations without double-strand breakage. These advances have improved gene editing safety and precision, reducing unfavorable effects. Lipid nanoparticles, which use viral vectors, improve therapeutic cell and tissue targeting. In rare disorders, gene therapy may be possible with CRISPR-Cas clinical trials. CRISPR-Cas research is improving gene editing, delivery, and rare disease treatment.}, }
@article {pmid39824579, year = {2025}, author = {Puri, B and Kulkarni, YA and Gaikwad, AB}, title = {Advances in CRISPR-Cas systems for kidney diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {149-162}, doi = {10.1016/bs.pmbts.2024.07.020}, pmid = {39824579}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Kidney Diseases/genetics/therapy ; Animals ; Gene Editing ; }, abstract = {Recent advances in CRISPR-Cas systems have revolutionised the study and treatment of kidney diseases, including acute kidney injury (AKI), chronic kidney disease (CKD), diabetic kidney disease (DKD), lupus nephritis (LN), and polycystic kidney disease (PKD). CRISPR-Cas technology offers precise and versatile tools for genetic modification in monogenic kidney disorders such as PKD and Alport syndrome. Recent advances in CRISPR technology have also shown promise in addressing other kidney diseases like AKI, CKD, and DKD. CRISPR-Cas holds promise to edit genetic mutations underlying these conditions, potentially leading to more effective and long-lasting treatments. Furthermore, the adaptability of CRISPR-Cas systems allows for developing tailored therapeutic strategies that specifically target the genetic and molecular mechanisms contributing to different kidney diseases. Beyond DNA modifications, CRISPR-Cas technologies also enable editing noncoding RNA, such as lncRNAs and miRNAs, in kidney diseases. Despite these advancements, significant challenges persist, including delivery efficiency to specific kidney cells and potential off-target effects. However, the rapid progress in CRISPR-Cas technology suggests a transformative impact on the future management of kidney diseases, offering the potential for enhanced patient outcomes through personalised and precise therapeutic approaches. This chapter highlights the recent advancement of CRISPR-Cas systems and their potential applications in various kidney diseases.}, }
@article {pmid39824578, year = {2025}, author = {Ajaykumar, CB and Rajkumar, S and Suresh, B and Birappa, G and Gowda, DAA and Jayachandran, A and Kim, KS and Hong, SH and Ramakrishna, S}, title = {Advances in applications of the CRISPR/Cas9 system for respiratory diseases.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {127-147}, doi = {10.1016/bs.pmbts.2024.07.014}, pmid = {39824578}, issn = {1878-0814}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Respiratory Tract Diseases/genetics/therapy ; Gene Editing ; Genetic Therapy/methods ; }, abstract = {Genetic and environmental factors can have an impact on lung and respiratory disorders which are associated with severe symptoms and have high mortality rates. Many respiratory diseases are significantly influenced by genetic or epigenetic factors. Gene therapy offers a powerful approach providing therapeutic treatment for lung diseases. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) are promising gene modifying tool that can edit the genome. The utilization of CRISPR/Cas9 systems in the investigation of respiratory disorders has resulted in advancements such as the rectification of deleterious mutations in patient-derived cells and the alteration of genes in multiple mammalian lung disease models. New avenues of treatment for lung disorders have been opened up by advances in CRISPR/Cas9 research. In this chapter, we discuss the known genes and mutations that cause several common respiratory disorders such as COPD, asthma, IPF, and ARDS. We further review the current research using CRISPR/Cas9 in numerous respiratory disorders and possible therapeutic treatments.}, }
@article {pmid39824577, year = {2025}, author = {Arora, Y and Priya, and Kumar, M and Kumar, D}, title = {Current approaches in CRISPR-Cas system for metabolic disorder.}, journal = {Progress in molecular biology and translational science}, volume = {210}, number = {}, pages = {1-19}, doi = {10.1016/bs.pmbts.2024.07.016}, pmid = {39824577}, issn = {1878-0814}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Metabolic Diseases/therapy/genetics ; Animals ; Gene Editing ; Genetic Therapy/methods ; }, abstract = {A new era in genomic medicine has been brought by the development of CRISPR-Cas technology, which presents hitherto unheard-of possibilities for the treatment of metabolic illnesses. The treatment approaches used in CRISPR/Cas9-mediated gene therapy, emphasize distribution techniques such as viral vectors and their use in preclinical models of metabolic diseases like hypercholesterolemia, glycogen storage diseases, and phenylketonuria. The relevance of high-throughput CRISPR screens for target identification in discovering new genes and pathways associated with metabolic dysfunctions is an important aspect of the discovery of new approaches. With cutting-edge options for genetic correction and cellular regeneration, the combination of CRISPR-Cas technology with stem cell therapy has opened new avenues for the treatment of metabolic illnesses. The integration of stem cell therapy and CRISPR-Cas technology is an important advance in the treatment of metabolic diseases, which are difficult to treat because of their intricate genetic foundations. This chapter addresses the most recent developments in the application of stem cell therapy and CRISPR-Cas systems to treat a variety of metabolic disorders, providing fresh hope for effective and maybe curative therapies. This chapter examines techniques and developments that have been made recently to address a variety of metabolic disorders using CRISPR-Cas systems. Our chapter focuses on the foundational workings of CRISPR-Cas technology and its potential uses in gene editing, gene knockout, and activation/repression-based gene modification.}, }
@article {pmid39626660, year = {2025}, author = {Jensen, CL and Chen, LF and Swigut, T and Crocker, OJ and Yao, D and Bassik, MC and Ferrell, JE and Boettiger, AN and Wysocka, J}, title = {Long-range regulation of transcription scales with genomic distance in a gene-specific manner.}, journal = {Molecular cell}, volume = {85}, number = {2}, pages = {347-361.e7}, doi = {10.1016/j.molcel.2024.10.021}, pmid = {39626660}, issn = {1097-4164}, support = {R35 GM131792/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; Mice ; *Promoter Regions, Genetic ; *Mouse Embryonic Stem Cells/metabolism/cytology ; Transcription, Genetic ; Chromatin/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; Enhancer Elements, Genetic ; Transcriptional Activation ; Gene Expression Regulation ; Transcription Factors/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {Although critical for tuning the timing and level of transcription, enhancer communication with distal promoters is not well understood. Here, we bypass the need for sequence-specific transcription factors (TFs) and recruit activators directly using a chimeric array of gRNA oligos to target dCas9 fused to the activator VP64-p65-Rta (CARGO-VPR). We show that this approach achieves effective activator recruitment to arbitrary genomic sites, even those inaccessible when targeted with a single guide. We utilize CARGO-VPR across the Prdm8-Fgf5 locus in mouse embryonic stem cells (mESCs), where neither gene is expressed. Although activator recruitment to any tested region results in the transcriptional induction of at least one gene, the expression level strongly depends on the genomic distance between the promoter and activator recruitment site. However, the expression-distance relationship for each gene scales distinctly in a manner not attributable to differences in 3D contact frequency, promoter DNA sequence, or the presence of repressive chromatin marks at the locus.}, }
@article {pmid39585363, year = {2025}, author = {Guo, Y and Zhao, X}, title = {CRISPR-based genetic screens in human pluripotent stem cells derived neurons and brain organoids.}, journal = {Cell and tissue research}, volume = {399}, number = {1}, pages = {1-8}, pmid = {39585363}, issn = {1432-0878}, support = {P50HD105353//National Institute of Child Health and Human Development/ ; R01 MH136152/MH/NIMH NIH HHS/United States ; R01 NS138268/NS/NINDS NIH HHS/United States ; P50 HD105353/HD/NICHD NIH HHS/United States ; R01 MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; R01MH118827/MH/NIMH NIH HHS/United States ; }, mesh = {Humans ; *Organoids/cytology/metabolism ; *Pluripotent Stem Cells/cytology/metabolism ; *Neurons/cytology/metabolism ; *Brain/cytology ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Testing/methods ; }, abstract = {Recent large-scale genome-wide association and single-cell RNA sequencing (scRNA-seq) studies have uncovered disease-associated genetic risk factors and cell type-specific genetic alterations. However, our understanding of how these genetic variants cause diseases and the underlying mechanisms remains largely unknown. Functional genomics screens using CRISPR-based technologies offer an effective tool for studying genes relevant to disease phenotypes. Here, we summarize recent CRISPR-based functional genomics screen approaches applied to human pluripotent stem cell (hPSC)-derived neurons and brain organoids. These screens have identified genes crucial for neurogenesis, neuronal survival, morphological development, and migration. Combining CRISPR-based genetic screens with scRNA-seq, researchers have revealed downstream genes and cellular pathways impacted by these genetic variants in human neural cells, providing new insights into the pathogenesis of neurodevelopmental disorders, such as microcephaly and autism spectrum disorders. Finally, we discuss current challenges and future directions for using CRISPR-based screens in furthering our understanding of neurological diseases and developing potential therapeutic strategies. Despite challenges, CRISPR-based screens have enormous potential for advancing the therapeutic development of many diseases.}, }
@article {pmid39822055, year = {2025}, author = {Göritzer, K and Melnik, S and Schwestka, J and Arcalis, E and Drapal, M and Fraser, P and Ma, JK and Stoger, E}, title = {Enhancing quality and yield of recombinant secretory IgA antibodies in Nicotiana benthamiana by endoplasmic reticulum engineering.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.14576}, pmid = {39822055}, issn = {1467-7652}, support = {760331//Horizon 2020 Framework Programme/ ; 21EBTA/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; J4583//Austrian Science Fund/ ; W1224//Austrian Science Fund/ ; }, abstract = {The production of complex multimeric secretory immunoglobulins (SIgA) in Nicotiana benthamiana leaves is challenging, with significant reductions in complete protein assembly and consequently yield, being the most important difficulties. Expanding the physical dimensions of the ER to mimic professional antibody-secreting cells can help to increase yields and promote protein folding and assembly. Here, we expanded the ER in N. benthamiana leaves by targeting the enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyses the rate-limiting step in the synthesis of the key membrane component phosphatidylcholine (PC). We used CRISPR/Cas to perform site-directed mutagenesis of each of the three endogenous CCT genes in N. benthamiana by introducing frame-shifting indels to remove the auto-inhibitory C-terminal domains. We generated stable homozygous lines of N. benthamiana containing different combinations of the edited genes, including plants where all three isofunctional CCT homologues were modified. Changes in ER morphology in the mutant plants were confirmed by in vivo confocal imaging and substantially increased the yields of two fully assembled SIgAs by prolonging the ER residence time and boosting chaperone accumulation. Through a combination of ER engineering with chaperone overexpression, we increased the yields of fully assembled SIgA by an order of magnitude, reaching almost 1 g/kg fresh leaf weight. This strategy removes a major roadblock to producing SIgA and will likely facilitate the production of other complex multimeric biopharmaceutical proteins in plants.}, }
@article {pmid39821831, year = {2025}, author = {Xie, Y and Liu, X and Wu, T and Luo, Y}, title = {Harnessing the Streptomyces-originating type I-E CRISPR/Cas system for efficient genome editing in Streptomyces.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, pmid = {39821831}, issn = {1869-1889}, abstract = {Since their discovery, CRISPR/Cas systems have significantly expanded the genetic toolbox, aiding in the exploration and enhanced production of natural products across various microbes. Among these, class 2 CRISPR/Cas systems are simpler and more broadly used, but they frequently fail to function effectively in many Streptomyces strains. In this study, we present an engineered class 1 type I CRISPR/Cas system derived from Streptomyces avermitilis, which enables efficient gene editing in phylogenetically distant Streptomyces strains. Through a plasmid interference assay, we identified the effective protospacer adjacent motif as 5'-AAN-3'. Utilizing this system, we achieved targeted chromosomal deletions ranging from 8 bp to 100 kb, with efficiencies exceeding 92%. We further utilized this system to insert DNA fragments into different Streptomyces genomes, facilitating the heterologous expression of exogenous genes and the activation of endogenous natural product biosynthetic gene clusters. Overall, we established a type I CRISPR/Cas-based gene-editing methodology that significantly advances the exploration of Streptomyces, known for their rich natural product resources. This is the first report of a gene editing tool developed based on the endogenous class 1 type I CRISPR/Cas system in Streptomyces spp. Our work enriches the Streptomyces gene manipulation toolbox and advances the discovery of valuable natural products within these organisms.}, }
@article {pmid39821024, year = {2025}, author = {Bu, W and Li, Y}, title = {Rat Models of Breast Cancer.}, journal = {Advances in experimental medicine and biology}, volume = {1464}, number = {}, pages = {123-148}, pmid = {39821024}, issn = {0065-2598}, mesh = {Animals ; Female ; Rats ; Humans ; *CRISPR-Cas Systems ; Breast Neoplasms/genetics/pathology/therapy ; Disease Models, Animal ; Mammary Neoplasms, Experimental/genetics/pathology ; Gene Editing/methods ; Mammary Glands, Animal/pathology/metabolism ; }, abstract = {As the first mammal to be domesticated for research purposes, rats served as the primary animal model for various branches of biomedical research, including breast cancer studies, up until the late 1990s and early 2000s. During this time, genetic engineering of mice, but not rats, became routine, and mice gradually supplanted rats as the preferred rodent model. But recent advances in creating genetically engineered rat models, especially with the assistance of CRISPR/Cas9 technology, have rekindled the significance of rats as a critical model in exploring various facets of breast cancer research. This is particularly pronounced in the study of the formation and progression of the estrogen receptor-positive subtype, which remains challenging to model in mice. In this chapter, we embark on a historical journey through the evolution of rat models in biomedical research and provide an overview of the general and histological characteristics of rat mammary glands. Next, we critically review major rat models for breast cancer research, including those induced by carcinogens, hormones, radiation, germline transgenes, germline knockouts, and intraductal injection of retrovirus/lentivirus to deliver oncogenic drivers into mature mammary glands. We also discuss the advances in building rat models using somatic genome editing powered by CRISPR/Cas9. This chapter concludes with our forward-looking perspective on future applications of advanced rat models in critical areas of breast cancer research that have continued to challenge the mouse model community.}, }
@article {pmid39820420, year = {2025}, author = {Dong, Y and Zhu, J and Pan, N}, title = {Recent advances in rapid detection of Helicobacter pylori by lateral flow assay.}, journal = {Archives of microbiology}, volume = {207}, number = {2}, pages = {35}, pmid = {39820420}, issn = {1432-072X}, mesh = {*Helicobacter pylori/isolation & purification/genetics ; *Helicobacter Infections/diagnosis/microbiology ; Humans ; Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; Point-of-Care Systems ; Enzyme-Linked Immunosorbent Assay/methods ; }, abstract = {Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy.}, }
@article {pmid39819332, year = {2025}, author = {Wang, C and Zhou, Y and Wang, Y and Jiao, P and Liu, S and Guan, S and Ma, Y}, title = {CRISPR-Cas9-mediated editing of ZmPL1 gene improves tolerance to drought stress in maize.}, journal = {GM crops & food}, volume = {16}, number = {1}, pages = {1-16}, doi = {10.1080/21645698.2024.2448869}, pmid = {39819332}, issn = {2164-5701}, mesh = {*Zea mays/genetics/physiology ; *Plants, Genetically Modified/genetics ; *Droughts ; *Plant Proteins/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Stress, Physiological/genetics ; Gene Expression Regulation, Plant ; Germination/genetics ; }, abstract = {Maize (Zea mays L.) is a widely grown food crop around the world. Drought stress seriously affects the growth and development process of plants and causes serious damage to maize yield. In the early stage, our research group conducted transcriptome sequencing analysis on the drought-resistant maize inbred line H8186 and screened out a gene with significantly down-regulated expression, Phylloplanin-like (ZmPL1). The ZmPL1 gee expression pattern was analyzed under various abiotic stresses, and the results showed that this gene was greatly affected by drought stress. Subcellular localization analysis showed that the protein was localized on the cell membrane. In order to verify the role of ZmPL1 in drought stress, we overexpressed ZmPL1 in yeast and found that the expression of ZmPL1 could significantly increase the drought sensitivity of yeast. Next, ZmPL1 transgenic plants were obtained by infecting maize callus using Agrobacterium-mediated method. Under drought stress, compared with overexpression lines, gene-edited lines had higher germination rate and seedling survival rate, lower accumulation of MDA, relative conductivity and ROS, higher antioxidant enzyme activity, and the expression levels of stress-related genes and ROS scavenging-related genes were significantly increased. Exogenous application of ABA to each lines under drought stress attenuated the damage caused by drought stress on ZmPL overexpressing plants. In summary, ZmPL1 negatively regulates drought tolerance in maize.}, }
@article {pmid39818894, year = {2025}, author = {Kaiser, J}, title = {'Safe harbor' gene therapy approach may have first success.}, journal = {Science (New York, N.Y.)}, volume = {387}, number = {6731}, pages = {234-235}, doi = {10.1126/science.adv9833}, pmid = {39818894}, issn = {1095-9203}, mesh = {Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Infant ; Liver ; CRISPR-Cas Systems ; Agammaglobulinemia ; Severe Combined Immunodeficiency ; }, abstract = {An obscure gene editor was used to restore a missing liver enzyme in an infant with a devastating metabolic condition.}, }
@article {pmid39746849, year = {2025}, author = {Paialunga, E and Bagheri, N and Rossetti, M and Fabiani, L and Micheli, L and Chamorro-Garcia, A and Porchetta, A}, title = {Leveraging Synthetic Antibody-DNA Conjugates to Expand the CRISPR-Cas12a Biosensing Toolbox.}, journal = {ACS synthetic biology}, volume = {14}, number = {1}, pages = {171-178}, doi = {10.1021/acssynbio.4c00541}, pmid = {39746849}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *DNA/genetics ; *SARS-CoV-2/genetics/immunology ; Humans ; *Spike Glycoprotein, Coronavirus/genetics/immunology ; Antibodies/immunology ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology/genetics ; Saliva ; Endodeoxyribonucleases/genetics/metabolism ; Immunoassay/methods ; Limit of Detection ; Enzyme-Linked Immunosorbent Assay/methods ; Bacterial Proteins/genetics ; }, abstract = {We report here the use of antibody-DNA conjugates (Ab-DNA) to activate the collateral cleavage activity of the CRISPR-Cas12a enzyme. Our findings demonstrate that Ab-DNA conjugates effectively trigger the collateral cleavage activity of CRISPR-Cas12a, enabling the transduction of antibody-mediated recognition events into fluorescence outputs. We developed two different immunoassays using an Ab-DNA as activator of Cas12a: the CRISPR-based immunosensing assay (CIA) for detecting SARS-CoV-2 spike S protein, which shows superior sensitivity compared with the traditional enzyme-linked immunosorbent assay (ELISA), and the CRISPR-based immunomagnetic assay (CIMA). Notably, CIMA successfully detected the SARS-CoV-2 spike S protein in undiluted saliva with a limit of detection (LOD) of 890 pM in a 2 h assay. Our results underscore the benefits of integrating Cas12a-based signal amplification with antibody detection methods. The potential of Ab-DNA conjugates, combined with CRISPR technology, offers a promising alternative to conventional enzymes used in immunoassays and could facilitate the development of versatile CRISPR analytical platforms for the detection of non-nucleic acid targets.}, }
@article {pmid39623867, year = {2025}, author = {Giorgetti, OB and Haas-Assenbaum, A and Boehm, T}, title = {Probing TCR Specificity Using Artificial In Vivo Diversification of CDR3 Regions.}, journal = {European journal of immunology}, volume = {55}, number = {1}, pages = {e202451434}, doi = {10.1002/eji.202451434}, pmid = {39623867}, issn = {1521-4141}, support = {//Max Planck Society/ ; 256073931-SFB1160//Deutsche Forschungsgemeinschaft/ ; }, mesh = {Animals ; *Complementarity Determining Regions/genetics/immunology ; Mice ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; CRISPR-Cas Systems ; T-Cell Antigen Receptor Specificity ; Mice, Transgenic ; T-Lymphocytes/immunology ; Receptors, Antigen, T-Cell/immunology/genetics ; }, abstract = {The T-cell receptor sequences expressed on cells recognizing a specific peptide in the context of a given MHC molecule can be explored for common features that might explain their antigen specificity. However, despite the development of numerous experimental and bioinformatic strategies, the specificity problem remains unresolved. To address the need for additional experimental paradigms, we report here on an in vivo experimental strategy designed to artificially diversify a transgenic TCR by CRISPR/Cas9-mediated mutagenesis of Tcra and Tcrb chain genes. In this system, an initially monoclonal repertoire of known specificity is converted into an oligoclonal pool of TCRs of altered antigen reactivity. Tracking the fate of individual clonotypes during the intrathymic differentiation process illuminates the strong selective pressures that shape the repertoire of naïve T cells. Sequence analyses of the artificially diversified repertoires identify key amino acid residues in the CDR3 regions required for antigen recognition, indicating that artificial diversification of well-characterized TCR transgene sequences helps to reduce the complexities of learning the rules of antigen recognition.}, }
@article {pmid39818188, year = {2025}, author = {Liu, Z and Liu, Y and Zhang, S and Wen, Y and Wang, X and Han, J and Yin, H}, title = {Crystal structure of the anti-CRISPR protein AcrIE7.}, journal = {Biochemical and biophysical research communications}, volume = {748}, number = {}, pages = {151315}, doi = {10.1016/j.bbrc.2025.151315}, pmid = {39818188}, issn = {1090-2104}, abstract = {Bacterial adaptive immunity, driven by CRISPR-Cas systems, protects against foreign nucleic acids from mobile genetic elements (MGEs), like bacteriophages. The type I-E CRISPR-Cas system employs the Cascade (CRISPR-associated complex for antiviral defense) complex for target DNA cleavage, guided by crRNA. Anti-CRISPR (Acr) proteins, such as AcrIE7, counteract this defense by inhibiting Cascade activity. In this study, we characterized and determined the structure of AcrIE7, a unique member of the AcrIE family, using X-ray crystallography under two distinct crystallization conditions, achieving resolutions of 2.05 Å and 2.68 Å, respectively. Topological analysis revealed that AcrIE7 consists of seven α-helices with two distinct charge regions, likely mediating its inhibitory interactions. Structural flexibility analysis revealed notable structural stability differences between the two crystallization conditions, indicating varying rigidity of the AcrIE7 protein under different conditions. Homology searches and AlphaFold predictions reinforced the unique nature of AcrIE7, which exhibits a novel fold, underscoring its distinct role within the AcrIE family. These findings enhance our understanding of Acr proteins and provide a theoretical foundation for developing CRISPR-based gene-editing regulatory tools.}, }
@article {pmid39817514, year = {2025}, author = {Jungfer, K and Moravčík, Š and Garcia-Doval, C and Knörlein, A and Hall, J and Jinek, M}, title = {Mechanistic determinants and dynamics of cA6 synthesis in type III CRISPR-Cas effector complexes.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1277}, pmid = {39817514}, issn = {1362-4962}, support = {ERC-CoG-820152/ERC_/European Research Council/International ; 51NF40-182880//SNSF/ ; ETH-24-16-2//ETH Zurich/ ; //University of Zurich/ ; }, mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Adenine Nucleotides/metabolism/biosynthesis ; Cryoelectron Microscopy ; Bacterial Proteins/metabolism/genetics ; Binding Sites ; Models, Molecular ; Second Messenger Systems ; }, abstract = {Type III clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems (type III CRISPR-Cas systems) use guide RNAs to recognize RNA transcripts of foreign genetic elements, which triggers the generation of cyclic oligoadenylate (cOA) second messengers by the Cas10 subunit of the type III effector complex. In turn, cOAs bind and activate ancillary effector proteins to reinforce the host immune response. Type III systems utilize distinct cOAs, including cyclic tri- (cA3), tetra- (cA4) and hexa-adenylates (cA6). However, the molecular mechanisms dictating cOA product identity are poorly understood. Here we used cryoelectron microscopy to visualize the mechanism of cA6 biosynthesis by the Csm effector complex from Enterococcus italicus (EiCsm). We show that EiCsm synthesizes oligoadenylate nucleotides in 3'-5' direction using a set of conserved binding sites in the Cas10 Palm domains to determine the size of the nascent oligoadenylate chain. Our data also reveal that conformational dynamics induced by target RNA binding results in allosteric activation of Cas10 to trigger oligoadenylate synthesis. Mutations of a key structural element in Cas10 perturb cOA synthesis to favor cA3 and cA4 formation. Together, these results provide comprehensive insights into the dynamics of cOA synthesis in type III CRISPR-Cas systems and reveal key determinants of second messenger product selectivity, thereby illuminating potential avenues for their engineering.}, }
@article {pmid39817512, year = {2025}, author = {Zhang, L and Qiu, X and Zhou, Y and Luo, Z and Zhu, L and Shao, J and Xie, M and Wang, H}, title = {A trigger-inducible split-Csy4 architecture for programmable RNA modulation.}, journal = {Nucleic acids research}, volume = {53}, number = {2}, pages = {}, doi = {10.1093/nar/gkae1319}, pmid = {39817512}, issn = {1362-4962}, support = {2023YFF1205400//Ministry of Science and Technology/ ; 32371498//National Natural Science Foundation of China/ ; 202209009//Westlake Laboratory of Life Sciences and Biomedicine/ ; 2024SSYS0007//'Pioneer' and 'Leading Goose' R&D Program of Zhejiang/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; RNA/metabolism/chemistry/genetics ; Endoribonucleases/metabolism/genetics ; Transcriptome ; }, abstract = {The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.}, }
@article {pmid39817421, year = {2025}, author = {Debaenst, S and Jarayseh, T and De Saffel, H and Bek, JW and Boone, M and Josipovic, I and Kibleur, P and Kwon, RY and Coucke, PJ and Willaert, A}, title = {Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, doi = {10.7554/eLife.100060}, pmid = {39817421}, issn = {2050-084X}, support = {FWO.OPR.2020.0023.01//Fonds Wetenschappelijk Onderzoek/ ; }, mesh = {*Zebrafish/genetics ; Animals ; Disease Models, Animal ; Bone Density/genetics ; Osteogenesis Imperfecta/genetics ; CRISPR-Cas Systems ; Phenotype ; Zebrafish Proteins/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; }, abstract = {Heritable fragile bone disorders (FBDs), ranging from multifactorial to rare monogenic conditions, are characterized by an elevated fracture risk. Validating causative genes and understanding their mechanisms remain challenging. We assessed a semi-high throughput zebrafish screening platform for rapid in vivo functional testing of candidate FBD genes. Six genes linked to severe recessive osteogenesis imperfecta (OI) and four associated with bone mineral density (BMD) from genome-wide association studies were analyzed. Using CRISPR/Cas9-based crispant screening in F0 mosaic founder zebrafish, Next-generation sequencing confirmed high indel efficiency (mean 88%), mimicking stable knock-out models. Skeletal phenotyping at 7, 14, and 90 days post-fertilization (dpf) using microscopy, Alizarin Red S staining, and microCT was performed. Larval crispants showed variable osteoblast and mineralization phenotypes, while adult crispants displayed consistent skeletal defects, including malformed neural and haemal arches, vertebral fractures and fusions, and altered bone volume and density. In addition, aldh7a1 and mbtps2 crispants experienced increased mortality due to severe skeletal deformities. RT-qPCR revealed differential expression of osteogenic markers bglap and col1a1a, highlighting their biomarker potential. Our results establish zebrafish crispant screening as a robust tool for FBD gene validation, combining skeletal and molecular analyses across developmental stages to uncover novel insights into gene functions in bone biology.}, }
@article {pmid39817182, year = {2024}, author = {Xue, M and Gonzalez, DH and Osikpa, E and Gao, X and Lillehoj, PB}, title = {Rapid and automated interpretation of CRISPR-Cas13-based lateral flow assay test results using machine learning.}, journal = {Sensors & diagnostics}, volume = {}, number = {}, pages = {}, pmid = {39817182}, issn = {2635-0998}, abstract = {CRISPR-Cas-based lateral flow assays (LFAs) have emerged as a promising diagnostic tool for ultrasensitive detection of nucleic acids, offering improved speed, simplicity and cost-effectiveness compared to polymerase chain reaction (PCR)-based assays. However, visual interpretation of CRISPR-Cas-based LFA test results is prone to human error, potentially leading to false-positive or false-negative outcomes when analyzing test/control lines. To address this limitation, we have developed two neural network models: one based on a fully convolutional neural network and the other on a lightweight mobile-optimized neural network for automated interpretation of CRISPR-Cas-based LFA test results. To demonstrate proof of concept, these models were applied to interpret results from a CRISPR-Cas13-based LFA for the detection of the SARS-CoV-2 N gene, a key marker for COVID-19 infection. The models were trained, evaluated, and validated using smartphone-captured images of LFA devices in various orientations with different backgrounds, lighting conditions, and image qualities. A total of 3146 images (1569 negative, 1577 positive) captured using an iPhone 13 or Samsung Galaxy A52 Android smartphone were analyzed using the trained models, which classified the LFA results within 0.2 s with 96.5% accuracy compared to the ground truth. These results demonstrate the potential of machine learning to accurately interpret test results of CRISPR-Cas-based LFAs using smartphone-captured images in real-world settings, enabling the practical use of CRISPR-Cas-based diagnostic tools for self- and at-home testing.}, }
@article {pmid39816115, year = {2024}, author = {Syahrani, RA and Wanandi, SI and Arumsari, S and Nihayah, S and Watanabe, Y and Mizuno, S and Louisa, M and Wuyung, PE}, title = {Dual sgRNA-directed knockout survivin gene expression using CRISPR/Cas9 technology for editing survivin gene in triple-negative breast cancer.}, journal = {Narra J}, volume = {4}, number = {3}, pages = {e1177}, pmid = {39816115}, issn = {2807-2618}, mesh = {*Survivin/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Triple Negative Breast Neoplasms/genetics/therapy ; Cell Line, Tumor ; Female ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) offers a robust approach for genome manipulation, particularly in cancer therapy. Given its high expression in triple-negative breast cancer (TNBC), targeting survivin with CRISPR/Cas9 holds promise as a therapeutic strategy. The aim of this study was to design specific single guide ribonucleic acid (sgRNA) for CRISPR/Cas9 to permanently knock out the survivin gene, exploring its potential as a therapeutic approach in breast cancer while addressing potential off-target effects. Survivin gene knockout was conducted in the TNBC cell line BT549. Intron 1, exon 2, and intron 2 of the survivin gene were selected as sgRNA targets. These sgRNAs were designed in silico and then cloned into a CRISPR/Cas9 expression plasmid. The cleavage activity was assessed using an enhanced green fluorescent protein (EGFP) expression plasmid. The sgRNAs with higher cleavage activity were selected for the establishment of knockout cells. After transfecting the plasmid into the cells, the success of the survivin gene knockout was validated at the deoxyribonucleic acid (DNA) level using polymerase chain reaction (PCR) and sequencing analysis, and at the protein expression level using Western blotting. The study found that sgRNAs survin1A (targeting intron 1), survex2A (targeting intron 2), and survin2A (targeting intron 2) demonstrated higher cleavage activities compared to the other sgRNAs. However, using the single sgRNA, survex2A did not generate mutations in the survivin gene. At the protein level, survivin was still expressed, indicating that a single sgRNA was ineffective in knocking out the survivin gene. In contrast, the combination of sgRNA survin1A and sgRNA survin2A was more effective in generating mutations in the survivin gene, resulting in the deletion of the entire exon 2 and leading to a loss of survivin protein expression. In conclusion, our work provides specific sgRNAs and demonstrates the utilization of dual sgRNAs strategy in the CRISPR/Cas9 technology to knock out the survivin gene, showing potential in breast cancer therapy.}, }
@article {pmid39778581, year = {2025}, author = {Lin, DW and Zhang, L and Zhang, J and Chandrasekaran, S}, title = {Inferring metabolic objectives and trade-offs in single cells during embryogenesis.}, journal = {Cell systems}, volume = {16}, number = {1}, pages = {101164}, doi = {10.1016/j.cels.2024.12.005}, pmid = {39778581}, issn = {2405-4720}, mesh = {*Embryonic Development/physiology/genetics ; Animals ; *Single-Cell Analysis/methods ; Mice ; Embryonic Stem Cells/metabolism ; Machine Learning ; CRISPR-Cas Systems ; Cell Proliferation/physiology/genetics ; Humans ; Cell Cycle/physiology ; }, abstract = {While proliferating cells optimize their metabolism to produce biomass, the metabolic objectives of cells that perform non-proliferative tasks are unclear. The opposing requirements for optimizing each objective result in a trade-off that forces single cells to prioritize their metabolic needs and optimally allocate limited resources. Here, we present single-cell optimization objective and trade-off inference (SCOOTI), which infers metabolic objectives and trade-offs in biological systems by integrating bulk and single-cell omics data, using metabolic modeling and machine learning. We validated SCOOTI by identifying essential genes from CRISPR-Cas9 screens in embryonic stem cells, and by inferring the metabolic objectives of quiescent cells, during different cell-cycle phases. Applying this to embryonic cell states, we observed a decrease in metabolic entropy upon development. We further uncovered a trade-off between glutathione and biosynthetic precursors in one-cell zygote, two-cell embryo, and blastocyst cells, potentially representing a trade-off between pluripotency and proliferation. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39689711, year = {2025}, author = {Metzner, E and Southard, KM and Norman, TM}, title = {Multiome Perturb-seq unlocks scalable discovery of integrated perturbation effects on the transcriptome and epigenome.}, journal = {Cell systems}, volume = {16}, number = {1}, pages = {101161}, doi = {10.1016/j.cels.2024.12.002}, pmid = {39689711}, issn = {2405-4720}, mesh = {Humans ; *Transcriptome/genetics ; *Epigenome/genetics ; Transcription Factors/genetics/metabolism ; Chromatin/genetics ; Polycomb Repressive Complex 2/genetics/metabolism ; DNA-Binding Proteins/genetics/metabolism ; Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Cell Line ; Neoplasm Proteins/genetics ; Chromatin Assembly and Disassembly/genetics ; }, abstract = {Single-cell CRISPR screens link genetic perturbations to transcriptional states, but high-throughput methods connecting these induced changes to their regulatory foundations are limited. Here, we introduce Multiome Perturb-seq, extending single-cell CRISPR screens to simultaneously measure perturbation-induced changes in gene expression and chromatin accessibility. We apply Multiome Perturb-seq in a CRISPRi screen of 13 chromatin remodelers in human RPE-1 cells, achieving efficient assignment of sgRNA identities to single nuclei via an improved method for capturing barcode transcripts from nuclear RNA. We organize expression and accessibility measurements into coherent programs describing the integrated effects of perturbations on cell state, finding that ARID1A and SUZ12 knockdowns induce programs enriched for developmental features. Modeling of perturbation-induced heterogeneity connects accessibility changes to changes in gene expression, highlighting the value of multimodal profiling. Overall, our method provides a scalable and simply implemented system to dissect the regulatory logic underpinning cell state. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39528429, year = {2025}, author = {Ding, S and Zheng, J and Jia, C}, title = {DeepMEns: an ensemble model for predicting sgRNA on-target activity based on multiple features.}, journal = {Briefings in functional genomics}, volume = {24}, number = {}, pages = {}, doi = {10.1093/bfgp/elae043}, pmid = {39528429}, issn = {2041-2657}, support = {62071079//National Natural Science Foundation of China/ ; 3132024197//Fundamental Research Funds for the Central Universities/ ; JSKX202203//Hainan Normal University, Ministry of Education/ ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Deep Learning ; Gene Editing/methods ; Streptococcus pyogenes/genetics ; Humans ; }, abstract = {The CRISPR/Cas9 system developed from Streptococcus pyogenes (SpCas9) has high potential in gene editing. However, its successful application is hindered by the considerable variability in target efficiencies across different single guide RNAs (sgRNAs). Although several deep learning models have been created to predict sgRNA on-target activity, the intrinsic mechanisms of these models are difficult to explain, and there is still scope for improvement in prediction performance. To overcome these issues, we propose an ensemble interpretable model termed DeepMEns based on deep learning to predict sgRNA on-target activity. By using five different training and validation datasets, we constructed five sub-regressors, each comprising three parts. The first part uses one-hot encoding, wherein 0-1 representation of the secondary structure is used as the input to the convolutional neural network (CNN) with Transformer encoder. The second part uses the DNA shape feature matrix as the input to the CNN with Transformer encoder. The third part uses positional encoding feature matrices as the proposed input into a long short-term memory network with an attention mechanism. These three parts are concatenated through the flattened layer, and the final prediction result is the average of the five sub-regressors. Extensive benchmarking experiments indicated that DeepMEns achieved the highest Spearman correlation coefficient for 6 of 10 independent test datasets as compared to previous predictors, this finding confirmed that DeepMEns can accomplish state-of-the-art performance. Moreover, the ablation analysis also indicated that the ensemble strategy may improve the performance of the prediction model.}, }
@article {pmid39814846, year = {2025}, author = {Shin, SW and Kim, SH and Gasselin, A and Lee, GM and Lee, JS}, title = {Comprehensive genome-scale CRISPR knockout screening of CHO cells.}, journal = {Scientific data}, volume = {12}, number = {1}, pages = {71}, pmid = {39814846}, issn = {2052-4463}, support = {RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; }, mesh = {CHO Cells ; Animals ; *Cricetulus ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.}, }
@article {pmid39812375, year = {2025}, author = {Wang, C and Xu, X and Yao, W and Wang, L and Pang, X and Xu, S and Luo, X}, title = {Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.}, journal = {Nano letters}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.nanolett.4c05829}, pmid = {39812375}, issn = {1530-6992}, abstract = {CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.}, }
@article {pmid39810563, year = {2025}, author = {Wang, W and Du, H and Dai, C and Ma, H and Luo, S and Wang, X and Guo, M and Kong, D and Wei, D}, title = {Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.}, journal = {Nanoscale}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4nr03852e}, pmid = {39810563}, issn = {2040-3372}, abstract = {Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10[-18] M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.}, }
@article {pmid39774325, year = {2025}, author = {Funk, JS and Klimovich, M and Drangenstein, D and Pielhoop, O and Hunold, P and Borowek, A and Noeparast, M and Pavlakis, E and Neumann, M and Balourdas, DI and Kochhan, K and Merle, N and Bullwinkel, I and Wanzel, M and Elmshäuser, S and Teply-Szymanski, J and Nist, A and Procida, T and Bartkuhn, M and Humpert, K and Mernberger, M and Savai, R and Soussi, T and Joerger, AC and Stiewe, T}, title = {Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations.}, journal = {Nature genetics}, volume = {57}, number = {1}, pages = {140-153}, pmid = {39774325}, issn = {1546-1718}, support = {STI 182/15-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; STI 182/13-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2573//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 109546710 Project A10//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA 1923/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC1213 A10N//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JO 1473/1-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 031L0063//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; DZL//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; LOEWE iCANx//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 65-0004//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 66-LV06//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 71-0012//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 09 R/2018//José Carreras Leukämie-Stiftung (Deutsche José Carreras Leukämie-Stiftung)/ ; TACTIC//Deutsche Krebshilfe (German Cancer Aid)/ ; }, mesh = {Humans ; *Tumor Suppressor Protein p53/genetics ; *Gene Editing/methods ; *Mutagenesis ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation, Missense/genetics ; Cell Line, Tumor ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nonsense Mediated mRNA Decay/genetics ; }, abstract = {The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.}, }
@article {pmid39754554, year = {2025}, author = {Hu, F and Liu, K and Zhang, Y and Zhao, S and Zhang, T and Yao, C and Lv, X and Wang, J and Liu, X and Peng, N}, title = {Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {658-666}, doi = {10.1021/acs.analchem.4c05026}, pmid = {39754554}, issn = {1520-6882}, mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques ; *COVID-19/diagnosis/virology ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Point-of-Care Testing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Limit of Detection ; }, abstract = {Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.}, }
@article {pmid39725131, year = {2025}, author = {Compiro, P and Chomta, N and Nimnual, J and Sunantawanit, S and Payungporn, S and Rotcheewaphan, S and Keawsapsak, P}, title = {CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120101}, doi = {10.1016/j.cca.2024.120101}, pmid = {39725131}, issn = {1873-3492}, mesh = {*Mycobacterium/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.}, }
@article {pmid39720925, year = {2025}, author = {Xu, Y and Chen, B and He, M and Yuan, G and Hu, B}, title = {Dual-Amplification Single-Particle ICP-MS Strategy Based on Strand Displacement Amplification-CRISPR/Cas12a Amplification for Homogeneous Detection of miRNA.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {811-817}, doi = {10.1021/acs.analchem.4c05385}, pmid = {39720925}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Metal Nanoparticles/chemistry ; *Mass Spectrometry ; MCF-7 Cells ; Limit of Detection ; }, abstract = {MicroRNAs (miRNAs) regulate a myriad of biological processes and thus have been regarded as useful biomarkers in biomedical research and clinical diagnosis. The specific and highly sensitive detection of miRNAs is of significant importance. Herein, a sensitive and rapid dual-amplification elemental labeling single-particle inductively coupled plasma-mass spectrometry (spICP-MS) analytical method based on strand displacement amplification (SDA) and CRISPR/Cas12a was developed for miRNA-21 detection. Taking gold nanoparticles (AuNPs) as the elemental labels, the Au NP probe initially hybridized with linker DNA, forming large aggregates. In the absence of target miRNA-21, large aggregates of AuNPs will produce high pulse signals in spICP-MS detection. In the presence of the target miRNA-21, it triggered the SDA reaction, and the SDA products activated CRISPR/Cas12a's trans-cleavage activity to cleave the linker DNA, resulting in disassembly of the AuNP aggregates. The AuNP aggregates with smaller size displayed lower pulse signals in spICP-MS detection. Under the optimal conditions, a good relationship between the average pulse signal intensity of AuNP aggregates and the concentration of miRNA-21 was obtained in the range of 0.5 fmol L[-1]-100 pmol L[-1] with a quantification limit as low as 0.5 fmol L[-1]. The developed method was successfully used for determination of miRNA-21 in human breast cancer cell lines (SK-BR-3 and MCF-7) and real blood samples from breast cancer patients. It is versatile, can be adapted to detect other targets by modifying the specific sequence of the SDA template chain that is complementary to the analytes, and offers a promising strategy for detecting various biomarkers with high sensitivity and specificity.}, }
@article {pmid39716527, year = {2025}, author = {Liu, R and Ji, W and Jiang, M and Shen, J}, title = {CRISPR technology combined with isothermal amplification methods for the diagnosis of Candida albicans infection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120106}, doi = {10.1016/j.cca.2024.120106}, pmid = {39716527}, issn = {1873-3492}, mesh = {*Candida albicans/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Candidiasis/diagnosis/microbiology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-of-Care Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPR-Cas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 10[2] CFU/ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.}, }
@article {pmid39711046, year = {2025}, author = {Yu, H and Xu, PF and Liu, Y and Jia, ZS and Li, YY and Tang, HW}, title = {LRET-Based Simultaneous Detection of Dual miRNAs via Multitrap Optical Tweezers Assisted Suspension Array Tagged by Two Different Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {602-612}, doi = {10.1021/acs.analchem.4c04895}, pmid = {39711046}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Optical Tweezers ; *CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Fluorescence Resonance Energy Transfer ; Luminescence ; Nucleic Acid Amplification Techniques ; Limit of Detection ; MCF-7 Cells ; }, abstract = {Nowadays, optical tweezers play a vital role not only in optical manipulation but also in bioassay. As principal optical trapping objects, microbeads can combine optical tweezers with suspension array technology, with amply focused laser beams and adequately concentrated tags contributing to highly sensitive detection. In view of the inefficiency of conventional single-trap optical tweezers, multitrap systems are developed. Here, green- and blue-emitting core-shell-shell upconversion nanoparticles (UCNPs) are adopted to encode microbeads and determine dual miRNAs, with the internal shells leading the luminescence process to facilitate quenching through luminescence resonance energy transfer (LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence signals are recovered and amplified, causing further enhanced detection sensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM are obtained with excellent specificities verified. Furthermore, dual miRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected, with results consistent with those obtained by PCR. Notably, miR-155 in MCF-7 and A549 cells is detectable at the single-cell level. Thus, the differences in the measured miRNA levels between MCF-7 and MCF-10A cells imply the potential of this method to discriminate breast cancer cells from epithelial cells despite the difficulty in distinguishing different cancer cells due to similar miRNA levels.}, }
@article {pmid39704707, year = {2025}, author = {Deng, R and Sheng, J and Xie, Z and Yang, H and Yang, S and Xie, S and Tang, X and Zhao, S and Dong, H and Chen, M and Chang, K}, title = {miR-Cabiner: A Universal microRNA Sensing Platform Based on Self-Stacking Cascaded Bicyclic DNA Circuit-Mediated CRISPR/Cas12a.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {799-810}, doi = {10.1021/acs.analchem.4c05370}, pmid = {39704707}, issn = {1520-6882}, mesh = {*MicroRNAs/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Biosensing Techniques/methods ; }, abstract = {CRISPR/Cas12a-based diagnostics have great potential for sensing nucleic acids, but their application is limited by the sequence-dependent property. A platform termed miR-Cabiner (a universal miRNA sensing platform based on self-stacking cascaded bicyclic DNA circuit-mediated CRISPR/Cas12a) is demonstrated herein that is sensitive and universal for analyzing miRNAs. This platform combines catalytic hairpin assembly (CHA) and hybrid chain reaction (HCR) into a unified circuit and finally cascades to CRISPR/Cas12a. Compared with the CHA-Cas12a and HCR-Cas12a systems, miR-Cabiner exhibits a significantly higher reaction rate. Panels of miRNAs (miR-130a, miR-10b, miR-21, and miR-1285), which are associated with diagnosis, staging, and prognosis of breast cancer, are designed to demonstrate the universality of miR-Cabiner. Four miRNAs can be detected to the fM-level by simply tuning the sequence in CHA components. Additionally, miRNA panel analysis also shows high accuracy in practical samples. This universally applicable platform for detecting miRNA may serve as an excellent tool for clinical diagnosis.}, }
@article {pmid39701943, year = {2025}, author = {Zhao, Z and Xiong, Q and Zhu, Y and Zhang, C and Li, Z and Chen, Z and Zhang, Y and Deng, X and Tao, Y and Xu, S}, title = {CRISPR/Cas12a-Enabled Amplification-Free Colorimetric Visual Sensing Strategy for Point-of-Care Diagnostics of Biomarkers.}, journal = {Analytical chemistry}, volume = {97}, number = {1}, pages = {1019-1027}, doi = {10.1021/acs.analchem.4c06196}, pmid = {39701943}, issn = {1520-6882}, mesh = {Humans ; *Colorimetry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Gold/chemistry ; BRCA1 Protein/genetics ; Metal Nanoparticles/chemistry ; Point-of-Care Testing ; Biomarkers, Tumor/blood/analysis ; Benzidines/chemistry ; Point-of-Care Systems ; Horseradish Peroxidase/chemistry/metabolism ; Biomarkers/blood/analysis ; Hydrogen Peroxide/chemistry ; }, abstract = {CRISPR/Cas12a-based biosensors have garnered significant attention in the field of point-of-care testing (POCT), yet the majority of the CRISPR-based POCT methods employ fluorescent systems as report probes. Herein, we report a new CRISPR/Cas12a-enabled multicolor visual biosensing strategy for the rapid detection of disease biomarkers. The proposed assay provided vivid color responses to enhance the accuracy of visual detection. In the existence of the target, the trans-cleavage activity of CRISPR-Cas12a was activated. The report probe modified with magnetic beads (MBs) and horseradish peroxidase (HRP) was cleaved, and HRP was released in the supernatant. As a result, HRP mediated the etching of gold nanobipyramids (AuNBPs) under hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine and generated a vivid color response. The proposed method has been verified by the detection of the breast cancer 1 gene (BRCA1) as a proof-of-principle target. According to the different colors of AuNBPs, our experimental results have demonstrated that as low as 30 pM BRCA1 can be detected with no more than 60 min. Additionally, the proposed sensor has been successfully applied in the analysis of BRCA1 in human serum samples with satisfactory results, which indicates great potential for the sensitive determination of biomarkers and the POCT area.}, }
@article {pmid39670529, year = {2025}, author = {Chang, G and Li, Y and Peng, L and Shen, C and Lu, Y and Teng, W and Liu, Y and Wang, Y and Zhu, W and Liu, C and He, X and Tong, Y and Zhao, X}, title = {TavWA1 is critical for wheat growth by modulating cell morphology and arrangement.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {1}, pages = {71-86}, doi = {10.1111/jipb.13807}, pmid = {39670529}, issn = {1744-7909}, support = {2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; XDA24010202//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; }, mesh = {*Triticum/genetics/growth & development/metabolism ; *Plant Proteins/metabolism/genetics ; *Mutation/genetics ; Phenotype ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems/genetics ; }, abstract = {Plant growth is determined by the production of cells and initiation of new organs. Exploring genes that control cell number and cell size is of great significance for understanding plant growth regulation. In this study, we characterized two wheat mutants, ah and dl, with abnormal growth. The ah mutant is a naturally occurring variant characterized by severe dwarfism, increased tiller number, and reduced grain length, while the dl mutant is derived from an ethyl methane sulfonate (EMS)-mutagenized population and exhibits smaller grain size and slightly reduced plant height. Cytological analyses revealed abnormal cell number, cell morphology and arrangement in the stems and leaves of the ah mutant, along with reduced cell length in the grains of the dl mutant. Map-based cloning identified that both mutants carry mutations in the same gene TavWA1-7D, which encodes a protein with a von Willebrand factor A (vWA) domain. The ah mutant harbors a 174-bp insertion in the 1,402-bp coding sequence (CDS) of TavWA1-7D, causing premature termination of protein translation, while the dl mutant contains a Glu420Lys substitution. Mimicking the TavWA1-7D[ah] through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing leads to a severe dwarfism phenotype. The C-terminus of the protein is crucial for its correct subcellular localization and interaction, supporting its critical role for TavWA1-7D function. Proteomic analysis showed that the dwarf phenotype of the ah mutant is associated with impaired photosynthesis, ribosome function, and nucleosome formation. Additionally, TavWA1-7D interacts with an E3 ligase, TaVIP1-3B, the expression levels of which are elevated in both mutants. Overexpression and knockout studies of TaVIP1-3B demonstrated its negative regulatory role in cell length and grain size. Together, our findings suggest that TavWA1-7D plays a vital role in regulating wheat growth and yield-related traits, with the dl mutant's short grain phenotype being associated with TaVIP1-3B expression levels.}, }
@article {pmid39642875, year = {2025}, author = {Rottner, AK and Lundin, A and Li, S and Firth, M and Maresca, M and Sienski, G}, title = {Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.}, journal = {Stem cell reports}, volume = {20}, number = {1}, pages = {102372}, doi = {10.1016/j.stemcr.2024.11.002}, pmid = {39642875}, issn = {2213-6711}, mesh = {Humans ; *Alzheimer Disease/genetics ; *Gene Editing/methods ; *Apolipoprotein E4/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; }, abstract = {Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.}, }
@article {pmid39642091, year = {2025}, author = {Basol, M and Ersoz-Gulseven, E and Ozaktas, H and Kalyoncu, S and Utine, CA and Cakan-Akdogan, G}, title = {Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.}, journal = {The FEBS journal}, volume = {292}, number = {2}, pages = {373-390}, doi = {10.1111/febs.17337}, pmid = {39642091}, issn = {1742-4658}, support = {219S943//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; }, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Carbohydrate Sulfotransferases ; *Sulfotransferases/genetics/metabolism ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Phenotype ; *Disease Models, Animal ; Cornea/pathology/metabolism ; CRISPR-Cas Systems ; Keratan Sulfate/genetics/metabolism ; Humans ; Gene Editing ; }, abstract = {The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.}, }
@article {pmid39471459, year = {2025}, author = {Hesami, M and Pepe, M and Spitzer-Rimon, B and Eskandari, M and Jones, AMP}, title = {Epigenetic factors related to recalcitrance in plant biotechnology.}, journal = {Genome}, volume = {68}, number = {}, pages = {1-11}, doi = {10.1139/gen-2024-0098}, pmid = {39471459}, issn = {1480-3321}, mesh = {*Epigenesis, Genetic ; *Biotechnology/methods ; *Plant Somatic Embryogenesis Techniques/methods ; *Gene Editing/methods ; Plants/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; }, abstract = {This review explores the challenges and potential solutions in plant micropropagation and biotechnology. While these techniques have proven successful for many species, certain plants or tissues are recalcitrant and do not respond as desired, limiting the application of these technologies due to unattainable or minimal in vitro regeneration rates. Indeed, traditional in vitro culture techniques may fail to induce organogenesis or somatic embryogenesis in some plants, leading to classification as in vitro recalcitrance. This paper focuses on recalcitrance to somatic embryogenesis due to its promise for regenerating juvenile propagules and applications in biotechnology. Specifically, this paper will focus on epigenetic factors that regulate recalcitrance as understanding them may help overcome these barriers. Transformation recalcitrance is also addressed, with strategies proposed to improve transformation frequency. The paper concludes with a review of CRISPR-mediated genome editing's potential in modifying somatic embryogenesis-related epigenetic status and strategies for addressing transformation recalcitrance.}, }
@article {pmid39451161, year = {2025}, author = {Pan, W and Gao, C and Niu, D and Cheng, J and Zhang, J and Yan, X and Long, Q and Zhu, Y and Sun, W and Xie, Q and He, Y and Deng, XW and Zhang, H and Li, J}, title = {Efficient gene disruption in polyploid genome by Cas9-Trex2 fusion protein.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {1}, pages = {7-10}, doi = {10.1111/jipb.13797}, pmid = {39451161}, issn = {1744-7909}, support = {TSQN202103160//Taishan Scholar Foundation of Shandong Province/ ; ZR202211070163//the Key R&D Program of Shandong Province/ ; ZR2021ZD30//Natural Science Foundation of Shandong Province/ ; ZR2022ZD22//Natural Science Foundation of Shandong Province/ ; ZR202103010168//the Excellent Youth Foundation of Shandong Scientific Committee/ ; }, mesh = {*Polyploidy ; *Gene Editing/methods ; *Genome, Plant/genetics ; Triticum/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Exodeoxyribonucleases/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; }, abstract = {The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.}, }
@article {pmid39240004, year = {2025}, author = {Zhao, Y and Cheng, P and Liu, Y and Liu, C and Hu, Z and Xin, D and Wu, X and Yang, M and Chen, Q}, title = {A highly efficient soybean transformation system using GRF3-GIF1 chimeric protein.}, journal = {Journal of integrative plant biology}, volume = {67}, number = {1}, pages = {3-6}, doi = {10.1111/jipb.13767}, pmid = {39240004}, issn = {1744-7909}, support = {TD2022C003//Natural Science Foundation of Heilongjiang Province/ ; YQ2022C010//Natural Science Foundation of Heilongjiang Province/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U20A2027//National Natural Science Foundation of China/ ; 2021YFF1001202//National Key R&D Program of China/ ; 2023YFD2300101-02//National Key R&D Program of China/ ; }, mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; Recombinant Fusion Proteins/genetics/metabolism ; }, abstract = {Expression of GRF3-GIF1 chimera significantly enhanced regeneration and transformation efficiency in soybean, increasing the number of transformable cultivars. Moreover, GmGRF3-GIF1 can be combined with CRISPR/Cas9 for highly effective gene editing.}, }
@article {pmid39810095, year = {2025}, author = {Liu, W and Wang, D and He, Q and Cao, S and Cao, J and Zhao, H and Cui, J and Yang, F}, title = {A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {22}, pmid = {39810095}, issn = {1471-2180}, mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects ; Virulence/genetics ; *Virulence Factors/genetics ; *Klebsiella Infections/microbiology/prevention & control ; Animals ; *Biofilms/growth & development/drug effects ; *Bacterial Proteins/genetics/metabolism ; *Gene Deletion ; Gene Expression Regulation, Bacterial ; Moths/microbiology ; Larva/microbiology ; CRISPR-Cas Systems ; }, abstract = {The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.}, }
@article {pmid39810027, year = {2025}, author = {Weng, Y and Wang, Y and Wang, K and Wu, F and Wei, Y and Jiang, J and Zhu, Y and Wang, F and Xie, H and Xiao, Y and Cai, Q and Xie, H and Zhang, J}, title = {OsLOX1 positively regulates seed vigor and drought tolerance in rice.}, journal = {Plant molecular biology}, volume = {115}, number = {1}, pages = {16}, pmid = {39810027}, issn = {1573-5028}, support = {CARS-01-20//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation Project for High-quality Development and Surpasses of Agriculture between the Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2020R1023008//the Special Foundation of Non-Profit Research Institutes of Fujian Province/ ; }, mesh = {*Oryza/genetics/physiology/enzymology ; *Seeds/genetics/physiology/growth & development ; *Droughts ; *Gene Expression Regulation, Plant ; *Germination/genetics ; *Plant Proteins/genetics/metabolism ; *Lipoxygenase/genetics/metabolism ; *Stress, Physiological ; Hydrogen Peroxide/metabolism ; Malondialdehyde/metabolism ; Plants, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Antioxidants/metabolism ; Superoxide Dismutase/metabolism/genetics ; Catalase/metabolism/genetics ; Drought Resistance ; }, abstract = {The lipoxygenase (LOX) gene family is widely distributed in plants, and its activity is closely associated with seed viability and stress tolerance. In this study, we cloned the rice(Oryza sativa)lipoxygenase gene OsLOX1, a key participant in the 13-lipoxygenase metabolic pathway. Our primary focus was to investigate its role in mediating responses to drought stress and seed germination in rice. Histochemical staining and qPCR analysis indicated that the expression level of OsLOX1 was relatively high in leaves and early germinating seeds. Our findings revealed that mutant lines with CRISPR/Cas9-induced knockout of OsLOX1 exhibited reduced tolerance to drought stress compared with the wild-type. This was accompanied by elevated levels of H2O2 and malondialdehyde, and a decrease in the expression levels of genes associated with antioxidant enzymes. Furthermore, knockout of OsLOX1 reduced the longevity of rice seeds increased H2O2 and MDA levels, and decreased the activities of the antioxidant enzymes superoxide dismutase and catalase, compared with the wild-type. These findings demonstrated that OsLOX1 positively regulated rice seed vigor and drought stress.}, }
@article {pmid39809780, year = {2025}, author = {Park, SJ and Ju, S and Jung, WJ and Jeong, TY and Yoon, DE and Lee, JH and Yang, J and Lee, H and Choi, J and Kim, HS and Kim, K}, title = {Robust genome editing activity and the applications of enhanced miniature CRISPR-Cas12f1.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {677}, pmid = {39809780}, issn = {2041-1723}, support = {RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; HEK293 Cells ; Apoptosis/genetics ; Genetic Therapy/methods ; Cell Line, Tumor ; CRISPR-Associated Proteins/metabolism/genetics ; }, abstract = {With recent advancements in gene editing technology using the CRISPR/Cas system, there is a demand for more effective gene editors. A key factor facilitating efficient gene editing is effective CRISPR delivery into cells, which is known to be associated with the size of the CRISPR system. Accordingly, compact CRISPR-Cas systems derived from various strains are discovered, among which Un1Cas12f1 is 2.6 times smaller than SpCas9, providing advantages for gene therapy research. Despite extensive engineering efforts to improve Un1Cas12f1, the editing efficiency of Un1Cas12f1 is still shown to be low depending on the target site. To overcome this limitation, we develop enhanced Cas12f1 (eCas12f1), which exhibits gene editing activity similar to SpCas9 and AsCpf1, even in gene targets where previously improved Un1Cas12f1 variants showed low gene editing efficiency. Furthermore, we demonstrate that eCas12f1 efficiently induces apoptosis in cancer cells and is compatible with base editing and regulation of gene expression, verifying its high utility and applicability in gene therapy research.}, }
@article {pmid39809757, year = {2025}, author = {Zou, J and Jiang, M and Xiao, R and Sun, H and Liu, H and Peacock, T and Tu, S and Chen, T and Guo, J and Zhao, Y and Barclay, W and Xie, S and Zhou, H}, title = {GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {670}, pmid = {39809757}, issn = {2041-1723}, support = {32025036//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32430104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021CFA016//Natural Science Foundation of Hebei Province (Hebei Provincial Natural Science Foundation)/ ; }, mesh = {Animals ; *Influenza A Virus, H1N1 Subtype/genetics/metabolism ; Swine ; Humans ; *Virus Replication ; *Receptors, Virus/metabolism/genetics ; CRISPR-Cas Systems ; Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics ; Orthomyxoviridae Infections/virology/metabolism ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; Cell Line ; Receptors, Cell Surface/metabolism/genetics ; Mice ; }, abstract = {The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type α-2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conduct a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduces virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX is revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributes to the receptor binding adaption of EA H1N1 SIV to the α-2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to α-2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1N1 SIV.}, }
@article {pmid39809734, year = {2025}, author = {Mormile, BW and Yan, Y and Bauer, T and Wang, L and Rivero, RC and Carpenter, SCD and Danmaigona Clement, C and Cox, KL and Zhang, L and Ma, X and Wheeler, TA and Dever, JK and He, P and Bogdanove, AJ and Shan, L}, title = {Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {644}, pmid = {39809734}, issn = {2041-1723}, support = {2018-67013-28513//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2020-67013-41537//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 18-123TX//Cotton Incorporated (Cotton Inc.)/ ; IOS-2421016//National Science Foundation (NSF)/ ; }, mesh = {*Xanthomonas/pathogenicity/genetics/metabolism ; *Plant Diseases/microbiology ; *Transcription Activator-Like Effectors/metabolism/genetics ; *Gossypium/microbiology ; Virulence/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; Gene Editing ; Host-Pathogen Interactions ; Genome, Bacterial ; Gene Expression Profiling ; }, abstract = {Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.}, }
@article {pmid39807869, year = {2025}, author = {Sáenz, JS and Rios-Galicia, B and Seifert, J}, title = {Antiviral defense systems in the rumen microbiome.}, journal = {mSystems}, volume = {}, number = {}, pages = {e0152124}, doi = {10.1128/msystems.01521-24}, pmid = {39807869}, issn = {2379-5077}, abstract = {The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanisms. However, the diversity and prevalence of antiviral defense systems in complex communities are still unknown. We therefore investigated the diversity and abundance of viral defense systems in 3,038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defense systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defense systems in the genomes. However, the number of defense systems, defense system families, and system density varied widely from genome to genome. Additionally, the number of defense system per genome correlated positively with the number of defense system families and the genome size. Restriction modification, Abi, and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defense systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage-host interaction.IMPORTANCEPhages may act antagonistically at the cell level but have a mutualistic interaction at the microbiome level. This interaction shapes the structure of microbial communities and is mainly driven by the defense mechanism. However, the diversity of such mechanism is larger than previously thought. Because of that, we described the abundance and diversity of the antiviral defense system of a collection of genomes, metagenome-assembled genomes (MAGs) and isolates, from the rumen. While defense mechanisms seem to be prevalent among bacteria and archaea, only a few were common. This suggests that most of these defense mechanisms are not present in many rumen microbes but could be shared among different members of the microbial community. This is consistent with the "pan-immune system" model, which appears to be common across different environments.}, }
@article {pmid39807514, year = {2025}, author = {Saha, D and Panda, AK and Datta, S}, title = {Critical considerations and computational tools in plant genome editing.}, journal = {Heliyon}, volume = {11}, number = {1}, pages = {e41135}, pmid = {39807514}, issn = {2405-8440}, abstract = {Recent advances in genome editing tools and CRISPR-Cas technologies have enabled plant genome engineering reach new heights. The current regulatory exemptions for certain categories of genome edited products, such as those derived from SDN-1 and SDN-2, which are free of any transgene, have significantly accelerated genome editing research in a number of agricultural crop plants in different countries. Although CRISPR-Cas technology is becoming increasingly popular, it is still important to carefully consider a number of factors before planning and carrying conducting CRISPR-Cas studies. To attempt genome editing in a plant, a high-quality genome sequence and a repeatable tissue culture protocol for in vitro regeneration are essential. One of the most important steps in plant genome editing is the designing of a CRISPR construct, which involves selecting the appropriate Cas protein, sgRNA sequence, and appropriate regulatory sequence to trigger expression. Computational tools and algorithms play a crucial role in construct design and gRNA selection to minimize off-target effects and also to optimize their delivery techniques. Researchers may need to select appropriate software tools capable of analyzing post-editing detection of mutation events and other DNA sequence abnormalities to identify off-target effects. To fully fulfill the potential of plant genome editing, continued advances in computational biology are essential to meet the challenges it faces today.}, }
@article {pmid39806509, year = {2025}, author = {Allais-Bonnet, A and Richard, C and André, M and Gelin, V and Deloche, MC and Lamadon, A and Morin, G and Mandon-Pépin, B and Canon, E and Thépot, D and Laubier, J and Moazami-Goudarzi, K and Laffont, L and Dubois, O and Fassier, T and Congar, P and Lasserre, O and Aguirre-Lavin, T and Vilotte, JL and Pailhoux, E}, title = {CRISPR/Cas9-editing of PRNP in Alpine goats.}, journal = {Veterinary research}, volume = {56}, number = {1}, pages = {11}, pmid = {39806509}, issn = {1297-9716}, support = {101000226//Horizon 2020 Framework Programme/ ; }, mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Female ; Goat Diseases/genetics ; Male ; Prion Proteins/genetics ; Gene Knockout Techniques/veterinary ; }, abstract = {Misfolding of the cellular PrP (PrP[c]) protein causes prion disease, leading to neurodegenerative disorders in numerous mammalian species, including goats. A lack of PrP[c] induces complete resistance to prion disease. The aim of this work was to engineer Alpine goats carrying knockout (KO) alleles of PRNP, the PrP[c]-encoding gene, using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the PRNP[Ter] mutation previously described in Norwegian goats. Genome editors were injected under the zona pellucida prior to the electroporation of 565 Alpine goat embryos/oocytes. A total of 122 two-cell-stage embryos were transferred to 46 hormonally synchronized recipient goats. Six of the goats remained pregnant and naturally gave birth to 10 offspring. Among the 10 newborns, eight founder animals carrying PRNP genome-edited alleles were obtained. Eight different mutated alleles were observed, including five inducing KO mutations. Three founders carried only genome-edited alleles and were phenotypically indistinguishable from their wild-type counterparts. Among them, one male carrying a one base pair insertion leading to a KO allele is currently used to rapidly extend a PRNP-KO line of Alpine goats for future characterization. In addition to KO alleles, a PRNP[del6] genetic variant has been identified in one-third of founder animals. This new variant will be tested for its potential properties with respect to prion disease. Future studies will also evaluate the effects of genetic background on other characters associated with PRNP KO, as previously described in the Norwegian breed or other species.}, }
@article {pmid39806441, year = {2025}, author = {Lin, Y and Li, C and Chen, Y and Gao, J and Li, J and Huang, C and Liu, Z and Wang, W and Zheng, X and Song, X and Wu, J and Wu, J and Luo, OJ and Tu, Z and Li, S and Li, XJ and Lai, L and Yan, S}, title = {RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington's disease models.}, journal = {Molecular neurodegeneration}, volume = {20}, number = {1}, pages = {4}, pmid = {39806441}, issn = {1750-1326}, mesh = {Animals ; *Huntington Disease/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Mice ; *Huntingtin Protein/genetics ; HEK293 Cells ; Swine ; Gene Editing/methods ; Mice, Transgenic ; }, abstract = {BACKGROUND: HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.
METHODS: The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.
RESULTS: CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.
CONCLUSIONS: These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.}, }
@article {pmid39516652, year = {2025}, author = {Murphy, R and Martin, KR}, title = {Genetic engineering and the eye.}, journal = {Eye (London, England)}, volume = {39}, number = {1}, pages = {57-68}, pmid = {39516652}, issn = {1476-5454}, mesh = {Humans ; *Genetic Therapy/methods ; *Genetic Engineering ; *Eye Diseases/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Vectors ; }, abstract = {The transformative potential of genetic engineering in ophthalmology is remarkable, promising new treatments for a wide range of blinding eye diseases. The eye is an attractive target organ for genetic engineering approaches, in part due to its relatively immune-privileged status, its accessibility, and the ease of monitoring of efficacy and safety. Consequently, the eye has been at the forefront of genetic engineering advances in recent years. The development of Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), base editors, prime editors, and transposases have enabled efficient and specific gene modification. Ocular gene therapy continues to progress, with recent advances in delivery systems using viral / non-viral vectors and novel promoters and enhancers. New strategies to achieve neuroprotection and neuroregeneration are evolving, including direct in-vivo cell reprogramming and optogenetic approaches. In this review, we discuss recent advances in ocular genetic engineering, examine their current therapeutic roles, and explore their potential use in future strategies to reduce the growing burden of vision loss and blindness.}, }
@article {pmid39806115, year = {2025}, author = {Zhang, X and Sun, R and Zheng, H and Qi, Y}, title = {Amplification-free sensitive detection of Staphylococcus aureus by spherical nucleic acid triggered CRISPR/Cas12a and Poly T-Cu reporter.}, journal = {Mikrochimica acta}, volume = {192}, number = {2}, pages = {76}, pmid = {39806115}, issn = {1436-5073}, support = {82073602//National Natural Science Foundation of China/ ; 20200201081JC//Natural Science Foundation of Jilin Province/ ; }, mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Copper/chemistry ; *Metal Nanoparticles/chemistry ; *Gold/chemistry ; *Limit of Detection ; Aptamers, Nucleotide/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; }, abstract = {A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus (S. aureus) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10[1]-1.0 × 10[6] CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases.}, }
@article {pmid39806080, year = {2025}, author = {Li, Y and Zeng, Z and Lv, X and Jiang, H and Li, A and Liu, Y and Deng, Y and Li, X}, title = {A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR/Cas12a.}, journal = {Mikrochimica acta}, volume = {192}, number = {2}, pages = {73}, pmid = {39806080}, issn = {1436-5073}, mesh = {*MicroRNAs/blood/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Testing ; *Limit of Detection ; Chorionic Gonadotropin/blood ; Nucleic Acid Hybridization ; Reagent Strips ; }, abstract = {A point-of-care testing (POCT) assay based on commercial HCG strip was proposed for miRNA21 detection by integrating RCA-HCR cascaded isothermal amplification with CRISPR/Cas12a. Three modules were integrated in the proposed platform: target amplification module composed of rolling circle amplification (RCA) cascaded with hybridization chain reaction (HCR), signal transduction module composed of CRISPR/Cas12a combined with HCG-agarose gel beads probes, and signal readout module composed of commercial HCG strips. The proposed RCA-HCR-CRISPR/Cas12a-HCG strip assay for miRNA21 detection had high sensitivity, and the limit of detection was as low as 37 fM. The proposed assay showed excellent specificity for miRNA21, as other miRNAs did not caused interference for detection. The recoveries of miRNA21 were ranged from 89.0 to 118.0%. The intra-batch and inter-batch coefficient of variation (CV) were 10.1-13.4% and 11.9-14.5%, respectively, which indicated a high accuracy and precision, and the serum matrix did not cause any interference. With the advantages of low-cost, high sensitivity, visualization, and easy popularization, the proposed assay is expected to provide a powerful tool for early diagnosis of tumor disease miRNA, especially in resource-limited areas.}, }
@article {pmid39804665, year = {2025}, author = {Liu, X and Huang, L and Li, M and Fu, Y and Zhang, W and Zhang, S and Liang, X and Shen, Q}, title = {An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2024.0057}, pmid = {39804665}, issn = {2573-1602}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T1 generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T0 generation sequencing results for genotype prediction. The T1 generation plants were then divided into two scenarios: ≥4 bp indels and 1-2 bp indels. Specific primers are designed for these categories, employing dual-primers critical annealing temperature PCR for ≥4 bp indels and the derived cleaved amplified polymorphic sequences (dCAPS) method for 1-2 bp indels. This method is straightforward, cost-effective, and allows rapid and precise identification of T1 editing outcomes, distinguishing between wild-type, heterozygous, and homozygous plants. This strategy accelerates gene functional analysis in plants and beyond.}, }
@article {pmid39804233, year = {2025}, author = {Wang, H and Zhao, R and Zhang, B and Xiao, Y and Yu, C and Wang, Y and Yu, C and Tang, Y and Li, Y and Lu, B and Li, B}, title = {Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON).}, journal = {Angewandte Chemie (International ed. in English)}, volume = {}, number = {}, pages = {e202423473}, doi = {10.1002/anie.202423473}, pmid = {39804233}, issn = {1521-3773}, abstract = {Solid-state nanopore is a promising single molecular detection technique, but is largely limited by relatively low resolution to small-size targets and laborious design of signaling probes. Here we establish a universal, CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON), which can accurately transduce different targeting sources/species into different DNA structural probes via a "Signal-ON" mode. Target recognition activates the cleavage activity of a Cas12a/crRNA system and then completely digest the blocker of an initiator. The unblocked initiator then triggers downstream DNA assembly reaction and generate a large-size structure easy for nanopore detection. Such integration of Cas12a/crRNA with DNA assembly establishes an accurate correspondence among the input targets, output DNA structures, and the ultimate nanopore signals. We demonstrated dsDNA, long RNA (i.e., Flu virus gene), short microRNA (i.e., let-7d) and non-nucleic acids (i.e., Pb2+) as input paradigms. Various structural assembly reactions, such as hybridization chain reaction (HCR), G-HCR and duplex polymerization strategy (DPS), are adapted as outputs for nanopore signaling. Simultaneous assay is also verified via transferring FluA and FluB genes into HCR and G-HCR, respectively. CANON is thus a modular sensing platform holding multiple advantages such as high accuracy, high resolution and high universality, which can be easily customized into various application scenes.}, }
@article {pmid39803585, year = {2024}, author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP}, title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39803585}, issn = {2692-8205}, abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.}, }
@article {pmid39803448, year = {2025}, author = {Hossain, KA and Nierzwicki, L and Orozco, M and Czub, J and Palermo, G}, title = {Flexibility in PAM Recognition Expands DNA Targeting in xCas9.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.08.26.609653}, pmid = {39803448}, issn = {2692-8205}, abstract = {xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.}, }
@article {pmid39801754, year = {2025}, author = {Guha, S and Jagadeesan, Y and Pandey, MM and Mittal, A and Chitkara, D}, title = {Targeting the epigenome with advanced delivery strategies for epigenetic modulators.}, journal = {Bioengineering & translational medicine}, volume = {10}, number = {1}, pages = {e10710}, pmid = {39801754}, issn = {2380-6761}, abstract = {Epigenetics mechanisms play a significant role in human diseases by altering DNA methylation status, chromatin structure, and/or modifying histone proteins. By modulating the epigenetic status, the expression of genes can be regulated without any change in the DNA sequence itself. Epigenetic drugs exhibit promising therapeutic efficacy against several epigenetically originated diseases including several cancers, neurodegenerative diseases, metabolic disorders, cardiovascular disorders, and so forth. Currently, a considerable amount of research is focused on discovering new drug molecules to combat the existing research gap in epigenetic drug therapy. A novel and efficient delivery system can be established as a promising approach to overcome the drawbacks associated with the current epigenetic modulators. Therefore, formulating the existing epigenetic drugs with distinct encapsulation strategies in nanocarriers, including solid lipid nanoparticles, nanogels, bio-engineered nanocarriers, liposomes, surface modified nanoparticles, and polymer-drug conjugates have been examined for therapeutic efficacy. Nonetheless, several epigenetic modulators are untouched for their therapeutic potential through different delivery strategies. This review provides a comprehensive up to date discussion on the research findings of various epigenetics mechanism, epigenetic modulators, and delivery strategies utilized to improve their therapeutic outcome. Furthermore, this review also highlights the recently emerged CRISPR tool for epigenome editing.}, }
@article {pmid39801562, year = {2025}, author = {Nguyen, T and Meleski, LWG and Belavatta, MP and Gurumoorthi, S and Zhang, C and Heins, AL and Zeng, AP}, title = {A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum.}, journal = {Engineering in life sciences}, volume = {25}, number = {1}, pages = {e202400026}, pmid = {39801562}, issn = {1618-0240}, abstract = {Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L[-1], and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.}, }
@article {pmid39787667, year = {2025}, author = {Hall, R and Sikora, T and Suter, A and Kuah, JY and Christodoulou, J and Van Bergen, NJ}, title = {Generation and heterozygous repair of human iPSC lines from two individuals with the neurodevelopmental disorder, TRAPPC4 deficiency.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103640}, doi = {10.1016/j.scr.2024.103640}, pmid = {39787667}, issn = {1876-7753}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodevelopmental Disorders/genetics ; *Heterozygote ; Male ; CRISPR-Cas Systems ; Female ; Cell Line ; Cell Differentiation ; }, abstract = {A rare neurodevelopmental disorder has been linked to a well-conserved splice site variant in the TRAPPC4 gene (c.454 + 3A > G), which causes mis-splicing of TRAPPC4 transcripts and reduced levels of TRAPPC4 protein. Patients present with severe progressive neurological symptoms including seizures, microcephaly, intellectual disability and facial dysmorphism. We have generated stem cells from fibroblasts of two individuals with the same homozygous TRAPPC4 c.454 + 3A > G pathogenic variant and used CRISPR/Cas9 editing to generate heterozygous gene-corrected isogenic controls. Clones were tested for pluripotency, differentiation potential, genotyped and karyotyped. These iPSC-based models will be used to understand disease mechanisms of TRAPPC4 disorder.}, }
@article {pmid39742627, year = {2025}, author = {Wei, S and Zhen, Y and Sun, C and Ma, Y and Li, Q and Wen, L}, title = {Generation of a USP9Y knockout human embryonic stem cell line with CRISPR-Cas9 technology.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103646}, doi = {10.1016/j.scr.2024.103646}, pmid = {39742627}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques/methods ; Male ; Ubiquitin Thiolesterase/genetics/metabolism ; Cell Line ; Cell Differentiation ; }, abstract = {Human embryonic stem cell (hESC) lines are vital tools for studying gene function, disease modeling, and therapy. We generated a USP9Y knockout hESC line using CRISPR-Cas9 in the male-derived H1 line. Targeted deletion of the USP9Y gene was confirmed via PCR and sequencing. The modified line retained pluripotency markers, exhibited a normal karyotype, and differentiated into all three germ layers. This model provides a valuable platform for studying USP9Y's role in human development and male infertility, offering insights into related disorders and therapeutic potential.}, }
@article {pmid39708407, year = {2025}, author = {Liu, X and Zhang, F and Chen, D and Yin, J and Bi, Z and Chen, L and Yan, J and Dong, Q and Peng, W and Xu, T and Guo, Y and Lin, H and Liu, H}, title = {Generation of INS-jGCaMP7f knock-in Ca[2+] reporter human embryonic stem cell line, GZLe001-C, using CRISPR/Cas9-based gene targeting.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103633}, doi = {10.1016/j.scr.2024.103633}, pmid = {39708407}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Calcium/metabolism ; Gene Targeting/methods ; Cell Line ; Gene Knock-In Techniques/methods ; Insulin/metabolism ; Genes, Reporter ; }, abstract = {As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca[2+] concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca[2+] level in response to different stimuli. This reporter cell line is a valuable research tool for studying functional maturation of hESC-derived insulin-producing cells, conducting drug screenings, and exploring the mechanisms of diabetes.}, }
@article {pmid39667064, year = {2025}, author = {Lu, S and Chen, M and Liu, X and Li, J and Liu, H and Li, S}, title = {Generation of a BEST1 Pr-EGFP reporter human embryonic stem cell line via CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103625}, doi = {10.1016/j.scr.2024.103625}, pmid = {39667064}, issn = {1876-7753}, mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Bestrophins/metabolism/genetics ; Retinal Pigment Epithelium/cytology/metabolism ; Genes, Reporter ; }, abstract = {The retinal pigment epithelium (RPE) cell, located between the neural retina and choriocapillaris, is vital for retinal maintenance and photoreceptor function. Human embryonic stem cells (hESCs) provide a limitless source of RPE cells for transplantation. Using CRISPR/Cas9, we inserted a fusion of the BEST1 promoter (an RPE-specific marker) and the EGFP gene into the AAVS1 locus to track differentiation in hESC-induced RPE (hESC-iRPE). The resulting gene-edited line, WAe009-A-2 M, maintained a normal karyotype, expressed pluripotency markers, and demonstrated differentiation potential, making it invaluable for RPE development and therapeutic research.}, }
@article {pmid39647515, year = {2025}, author = {Luo, X and Germer, J and Burghardt, T and Grau, M and Lin, Y and Höhn, M and Lächelt, U and Wagner, E}, title = {Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing.}, journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences}, volume = {205}, number = {}, pages = {106983}, doi = {10.1016/j.ejps.2024.106983}, pmid = {39647515}, issn = {1879-0720}, mesh = {*Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; HeLa Cells ; Hydrogen-Ion Concentration ; RNA, Guide, CRISPR-Cas Systems ; Peptides/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.}, }
@article {pmid39644600, year = {2025}, author = {Xu, Y and Wu, H and Jiang, J and Ye, L and Hao, K and Han, K and Hu, S and Lei, W and Guo, Z}, title = {Generation and characterization of the LINC01405 knockout human embryonic stem cell line.}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103619}, doi = {10.1016/j.scr.2024.103619}, pmid = {39644600}, issn = {1876-7753}, mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *RNA, Long Noncoding/genetics/metabolism ; *Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {Long Intergenic Non-Protein Coding RNA 1405 (LINC01405), with known elevated expression in muscle, has been linked to a number of musculo-skeletal conditions. By utilizing the CRISPR/Cas9 gene editing system, we generated a LINC01405 knockout human embryonic stem cell (hESC) line. This line remains human stem cell-like morphology and pluripotency, exhibits a normal karyotype, and can differentiate into cells from all three germ layers. This cell line will be an invaluable model for the research on LINC01405's role in normal development of cardiac and skeletal muscle, and their diseases.}, }
@article {pmid39380247, year = {2025}, author = {Tsuji-Hosokawa, A and Tsuchiya, I and Shimizu, K and Terao, M and Yasuhara, M and Miyamoto, N and Kikuchi, S and Ogawa, Y and Nakamura, K and Matsubara, Y and Takada, S}, title = {Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs.}, journal = {Journal of inherited metabolic disease}, volume = {48}, number = {1}, pages = {e12803}, doi = {10.1002/jimd.12803}, pmid = {39380247}, issn = {1573-2665}, support = {19ek0109290h0003//Japan Agency for Medical Research and Development/ ; 2023C-12//National Research Institute for Child Health and Development/ ; 20K18208//Japan Society for the Promotion of Science/ ; }, mesh = {*Phenylketonurias/genetics ; Animals ; *Gene Editing/methods ; *Disease Models, Animal ; Humans ; Mice ; Phenylalanine Hydroxylase/genetics ; Zygote ; Female ; Phenotype ; Male ; CRISPR-Cas Systems ; Genome, Human ; Phenylalanine/genetics ; }, abstract = {Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model Pah[hR111X] mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in Pah[hR111X] mice. Pah[hR111X] mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of Pah[hR111X] mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.}, }
@article {pmid39109936, year = {2025}, author = {Rose, JC and Belk, JA and Wong, IT and Luebeck, J and Horn, HT and Daniel, B and Jones, MG and Yost, KE and Hung, KL and Kolahi, KS and Curtis, EJ and Kuo, CJ and Bafna, V and Mischel, PS and Chang, HY}, title = {Disparate Pathways for Extrachromosomal DNA Biogenesis and Genomic DNA Repair.}, journal = {Cancer discovery}, volume = {15}, number = {1}, pages = {69-82}, doi = {10.1158/2159-8290.CD-23-1117}, pmid = {39109936}, issn = {2159-8290}, support = {K99-CA279512//National Cancer Institute (NCI)/ ; //Howard Hughes Medical Institute (HHMI)/ ; //A.P. Giannini Foundation/ ; CGCSDF-2021\100007//National Cancer Institute (NCI)/ ; CGCSDF-2021\100007//Cancer Research UK (CRUK)/ ; //Howard Hughes Medical Institute (HHMI)/ ; }, mesh = {Humans ; *DNA Repair ; DNA Breaks, Double-Stranded ; DNA, Circular/genetics ; DNA End-Joining Repair ; CRISPR-Cas Systems ; }, abstract = {Our study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between double-strand breaks. ecDNAs and their corresponding chromosomal scars can form via nonhomologous end joining or microhomology-mediated end joining, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.}, }
@article {pmid39800582, year = {2025}, author = {Zhang, Y and Dong, X and Jiang, C and Yu, Y and Zhang, H and Fu, J and Su, G and Liu, Y}, title = {Corrigendum to "A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples" [Talanta, Volume 286 (2025), 127491].}, journal = {Talanta}, volume = {}, number = {}, pages = {127558}, doi = {10.1016/j.talanta.2025.127558}, pmid = {39800582}, issn = {1873-3573}, }
@article {pmid39800499, year = {2025}, author = {Yu, F and Zhang, Q and Ma, T and Zhang, S and Wang, F and Yue, D and Liu, S and Liao, Y and Liu, LE and Wu, Y and Zang, W}, title = {Bifunctional probe propelling multipath strand displacement amplification tandem CRISPR/Cas12a for ultrasensitive and robust assay of DNA methyltransferase activity.}, journal = {Analytica chimica acta}, volume = {1337}, number = {}, pages = {343540}, doi = {10.1016/j.aca.2024.343540}, pmid = {39800499}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; DNA Methylation ; Limit of Detection ; DNA Modification Methylases/metabolism ; Enzyme Assays/methods ; DNA Probes/chemistry ; }, abstract = {BACKGROUND: DNA methylation catalyzed by various DNA methyltransferases (DNA MTases) is one of the important epigenetic regulations in both eukaryotes and prokaryotes. Therefore, the detection of DNA MTase activity is a vital target and direction in the study of methylation-related diseases.
RESULTS: In this study, an ultrasensitive and robust strategy was developed for DNA MTase activity sensing based on bifunctional probe propelling multipath strand displacement amplification and CRISPR/Cas12a techniques. First, a bifunctional hairpin probe (bHpDNA) was designed instead of a conventional single-function probe. In the presence of DNA MTase, the bHpDNA was methylated and cleaved by a restriction endonuclease into two independent primers, both of which bind with the templates to trigger strand displacement amplification and produce the active DNA of CRISPR/Cas12a. Second, annealing-assisted binding instead of free diffusion adhesion was used to improve hybridization efficiency between the primers and templates. Finally, the CRISPR/Cas12a system was used to achieve fluorescence signal output to analyze DNA MTase activity. If targets were absent, there was no signal because no primers were released from the bHpDNA. To verify the reliability of the method, two key DNA MTases, Dam and M. SssI, were analyzed, and their limits of detection were 2.458 × 10[-3] and 3.820 × 10[-3] U/mL, respectively, which were lower than those of most reported fluorescence methods.
SIGNIFICANCE: This method was successfully used in the evaluation of DNA MTase inhibitors and the detection of DNA MTase activity in complex biological systems with good recoveries and relative standard deviation at low spiked concentrations (0.1-1 U/mL), which all indicate that this method is an ultrasensitive and robust strategy in DNA MTase activity assay and has great potential in biomedical and clinical detection.}, }
@article {pmid39799111, year = {2025}, author = {Su-Tobon, Q and Fan, J and Goldstein, M and Feeney, K and Ren, H and Autissier, P and Wang, P and Huang, Y and Mohanty, U and Niu, J}, title = {CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {595}, pmid = {39799111}, issn = {2041-1723}, support = {1DP2HG011027-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; Graduate Research Fellowship//National Science Foundation (NSF)/ ; }, mesh = {*Aptamers, Nucleotide/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Directed Molecular Evolution/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; RNA-Binding Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.}, }
@article {pmid39798882, year = {2025}, author = {Kainov, DE and Ravlo, E and Ianevski, A}, title = {Seeking innovative concepts in development of antiviral drug combinations.}, journal = {Antiviral research}, volume = {}, number = {}, pages = {106079}, doi = {10.1016/j.antiviral.2025.106079}, pmid = {39798882}, issn = {1872-9096}, abstract = {Antiviral drugs are crucial for managing viral infections, but current treatment options remain limited, particularly for emerging viruses. These drugs can be classified based on their chemical composition, including neutralizing antibodies (nAbs), recombinant human receptors (rhRs), antiviral CRISPR/Cas systems, interferons, antiviral peptides (APs), antiviral nucleic acid polymers, and small molecules. Some of these agents target viral factors, host factors, or both. A major challenge for virus-targeted treatments is their narrow-spectrum effectiveness and the potential for drug resistance, while host-directed and virus-host-targeted therapies often suffer from significant side effects. The synergistic combination of multiple antiviral drugs holds promise for improving treatment outcomes by targeting different stages of the viral life cycle, reducing resistance, and minimizing side effects. However, developing such drug combinations presents its own set of challenges. Leveraging the accumulated knowledge, several drug combinations could be optimized, and new combinations developed, to more effectively treat both emerging and re-emerging viral infections.}, }
@article {pmid39798545, year = {2025}, author = {Yaremenko, AV and Khan, MM and Zhen, X and Tang, Y and Tao, W}, title = {Clinical advances of mRNA vaccines for cancer immunotherapy.}, journal = {Med (New York, N.Y.)}, volume = {6}, number = {1}, pages = {100562}, doi = {10.1016/j.medj.2024.11.015}, pmid = {39798545}, issn = {2666-6340}, mesh = {Humans ; *Cancer Vaccines/immunology/therapeutic use/administration & dosage ; *Neoplasms/therapy/immunology/genetics ; *mRNA Vaccines ; *Immunotherapy/methods ; RNA, Messenger/administration & dosage/genetics ; CRISPR-Cas Systems ; Antigens, Neoplasm/immunology/genetics ; Clinical Trials as Topic ; }, abstract = {The development of mRNA vaccines represents a significant advancement in cancer treatment, with more than 120 clinical trials to date demonstrating their potential across various malignancies, including lung, breast, prostate, melanoma, and more challenging cancers such as pancreatic and brain tumors. These vaccines work by encoding tumor-specific antigens and immune-stimulating molecules, effectively activating the immune system to target and eliminate cancer cells. Despite these promising advancements, significant challenges remain, particularly in achieving efficient delivery and precise regulation of the immune response. This review provides a comprehensive overview of recent clinical progress in mRNA cancer vaccines, discusses the innovative strategies being employed to overcome existing hurdles, and explores future directions, including the integration of CRISPR-Cas9 technology and advancements in mRNA design. Our aim is to provide insights into the ongoing research and clinical trials, highlighting the transformative potential of mRNA vaccines in advancing oncology and improving patient outcomes.}, }
@article {pmid39798420, year = {2025}, author = {Gao, Z and Liu, J and Zhang, Y and Xu, R and Yang, Y and Wu, L and Lei, J and Ming, T and Ren, F and Liu, L and Chen, Q}, title = {TdT combined with Cas14a for the electrochemical biosensing of NPC-derived exosomes.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {163}, number = {}, pages = {108900}, doi = {10.1016/j.bioelechem.2025.108900}, pmid = {39798420}, issn = {1878-562X}, abstract = {In this work, the electrochemical biosensor based on the subtle combination of terminal deoxynucleotidyl transferase (TdT), CRISPR/Cas14a, and magnetic nanoparticles (MNPs) was developed for the detection of nasopharyngeal carcinoma (NPC)-derived exosomes. Due to the synergistic effect of the following factors: the powerful elongation capacity of TdT for single-stranded DNA (ssDNA) with 3-hydroxy terminus, the outstanding trans-cleavage ability of CRISPR/Cas14a specifcally activated by the crRNA binding to target DNA, and the excellent separation ability of MNPs, the developed electrochemical biosensor exhibited high sensitivity for the detection of NPC-derived exosome, with a linear range from 6.0 × 10[2] ∼ 1.0 × 10[5] particles/mL and a limit of detection as lown as 80 particles/mL. In addition, this electrochemical biosensor successfully distinguished exosomes from NPC patients and healthy individuals. This electrochemical biosensor opens up a new pathway for the early diagnosis of NPC. Abbreviations: NPC, Nasopharyngeal carcinoma; CRISPR/Cas, Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system; PAMs, Protospacer adjacent motifs; RCA, Rolling circle amplification; CHA, Catalytic hairpin assembly; LAMP, Loop-mediated isothermal amplification; TdT, Terminal deoxynucleotidyl transferase; SgRNA, Single guide RNA.}, }
@article {pmid39562753, year = {2025}, author = {Cirincione, A and Simpson, D and Yan, W and McNulty, R and Ravisankar, P and Solley, SC and Yan, J and Lim, F and Farley, EK and Singh, M and Adamson, B}, title = {A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.}, journal = {Nature methods}, volume = {22}, number = {1}, pages = {92-101}, pmid = {39562753}, issn = {1548-7105}, support = {R35GM138167//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P30CA072720//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32HG003284//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32GM007388//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F31HD113443//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01-GM076275//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DGE-2039656//National Science Foundation (NSF)/ ; 2239957//National Science Foundation (NSF)/ ; }, mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Benchmarking ; HEK293 Cells ; }, abstract = {Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Here we assembled a prime editing platform capable of high-efficiency substitution editing suitable for functional interrogation of small genetic variants. We benchmarked this platform for pooled, loss-of-function screening using a library of ~240,000 engineered prime editing guide RNAs (epegRNAs) targeting ~17,000 codons with 1-3 bp substitutions. Comparing the abundance of these epegRNAs across screen samples identified negative selection phenotypes for 7,996 nonsense mutations targeted to 1,149 essential genes and for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Rigorous evaluation of codon-matched controls demonstrated that these phenotypes were highly specific to the intended edit. Altogether, we established a prime editing approach for multiplexed, functional characterization of genetic variants with simple readouts.}, }
@article {pmid39797940, year = {2025}, author = {Fu, Y and Zhang, P and Chen, F and Xie, Z and Xiao, S and Huang, Z and Lau, CH and Zhu, H and Luo, J}, title = {CRISPR detection of cardiac tumor-associated microRNAs.}, journal = {Molecular biology reports}, volume = {52}, number = {1}, pages = {114}, pmid = {39797940}, issn = {1573-4978}, support = {20212ACB206031//Jiangxi Provincial Natural Science Foundation/ ; 82260053//National Natural Science Foundation of China/ ; }, mesh = {Humans ; *MicroRNAs/genetics ; *Heart Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Myxoma/genetics/diagnosis ; Hemangiosarcoma/genetics/diagnosis ; Biomarkers, Tumor/genetics ; }, abstract = {As multiple imaging modalities cannot reliably diagnose cardiac tumors, the molecular approach offers alternative ways to detect rare ones. One such molecular approach is CRISPR-based diagnostics (CRISPR-Dx). CRISPR-Dx enables visual readout, portable diagnostics, and rapid and multiplex detection of nucleic acids such as microRNA (miRNA). Dysregulation of miRNA expressions has been associated with cardiac tumors such as atrial myxoma and angiosarcoma. Diverse CRISPR-Dx systems have been developed to detect miRNA in recent years. These CRISPR-Dx systems are generally classified into four classes, depending on the Cas proteins used (Cas9, Cas12, Cas13, or Cas12f). CRISPR/Cas systems are integrated with various isothermal amplifications to detect low-abundance miRNAs. Amplification-free CRISPR-Dx systems have also been recently developed to detect miRNA directly. Herein, we critically discuss the advances, pitfalls, and future perspectives for these CRISPR-Dx systems in detecting miRNA, focusing on the diagnosis and prognosis of cardiac tumors.}, }
@article {pmid39797776, year = {2025}, author = {Li, Q and Zhang, Z and Wu, X and Zhao, Y and Liu, Y}, title = {Cascade-Responsive Nanoparticles for Efficient CRISPR/Cas9-Based Glioblastoma Gene Therapy.}, journal = {ACS applied materials & interfaces}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsami.4c15671}, pmid = {39797776}, issn = {1944-8252}, abstract = {CRISPR/Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing technology represents great promise for treating glioblastoma (GBM) due to its potential to permanently eliminate tumor pathogenic genes. Unfortunately, delivering CRISPR to the GBM in a safe and effective manner is challenging. Herein, a glycosylated and cascade-responsive nanoparticle (GCNP) that can effectively cross the blood-brain barrier (BBB) and activate CRISPR/Cas9-based gene editing only in the GBM is designed. The GCNP possesses a cationic polyplex core and a glycosylated polymer layer that is capable of cascading response to low pH and high GSH concentration, so that the release of CRISPR/Cas9 only takes place after crossing the BBB and entering the GBM where the acidic tumor microenvironment and high concentration of glutathione (GSH) are present. By targeting the programmed death-ligand 1 (PD-L1) in GBM, GCNP effectively inhibited the tumor growth and greatly prolonged the survival time of GBM-bearing mice when combined with temozolomide (TMZ).}, }
@article {pmid39796169, year = {2025}, author = {Rallabandi, HR and Singh, MK and Looger, LL and Nath, SK}, title = {Defining Mechanistic Links Between the Non-Coding Variant rs17673553 in CLEC16A and Lupus Susceptibility.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, doi = {10.3390/ijms26010314}, pmid = {39796169}, issn = {1422-0067}, support = {5R01AI172255-02S1/NH/NIH HHS/United States ; }, mesh = {*Lectins, C-Type/genetics/metabolism ; Humans ; *Lupus Erythematosus, Systemic/genetics ; *Genetic Predisposition to Disease ; *Monosaccharide Transport Proteins/genetics ; *Polymorphism, Single Nucleotide ; Alleles ; Autophagy/genetics ; Genome-Wide Association Study ; Gene Editing ; STAT3 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems ; B-Lymphocytes/metabolism ; Chromosomes, Human, Pair 16/genetics ; }, abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus-particularly the risk allele-drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13.}, }
@article {pmid39795970, year = {2024}, author = {Cerna-Chavez, R and Ortega-Gasco, A and Baig, HMA and Ehrenreich, N and Metais, T and Scandura, MJ and Bujakowska, K and Pierce, EA and Garita-Hernandez, M}, title = {Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, doi = {10.3390/ijms26010114}, pmid = {39795970}, issn = {1422-0067}, support = {EY012910/EY/NEI NIH HHS/United States ; EY020902/EY/NEI NIH HHS/United States ; EY014104/NH/NIH HHS/United States ; Competitive Renewal Research Grant in Pediatric Ophthalmology//Knights Templar Eye Foundation/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Retinal Diseases/genetics/therapy ; Mutation ; }, abstract = {Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency.}, }
@article {pmid39795964, year = {2024}, author = {Tao, R and Zhang, J and Meng, L and Gao, J and Miao, C and Liu, W and Jin, W and Wan, Y}, title = {A Rapid Field-Visualization Detection Platform for Genetically Modified Soybean 'Zhonghuang 6106' Based on RPA-CRISPR.}, journal = {International journal of molecular sciences}, volume = {26}, number = {1}, pages = {}, doi = {10.3390/ijms26010108}, pmid = {39795964}, issn = {1422-0067}, support = {2022ZD0402003//Biological Breeding-Major Projects/ ; }, mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Gene Editing/methods ; }, abstract = {Genetically modified (GM) herbicide-tolerant soybean 'Zhonghuang 6106', which introduces a glyphosate-resistant gene, ensures soybean yield while allowing farmers to reduce the use of other herbicides, thereby reducing weed management costs. To protect consumer rights and facilitate government supervision, we have established a simple and rapid on-site nucleic acid detection method for GM soybean 'Zhonghuang 6106'. This method leverages the isothermal amplification characteristics of RPA technology and the high specificity of CRISPR-Cas12a to achieve high sensitivity and accuracy in detecting GM soybean components. By optimizing experimental conditions, the platform can quickly produce visual detection results, significantly reducing detection time and improving efficiency. The system can detect down to 10 copies/μL of 'Zhonghuang 6106' DNA templates, and the entire detection process takes about 1 h. The technology also has strong editing capabilities; by redesigning the primers and crRNA in the method, it can become a specific detection method for other GM samples, providing strong technical support for the regulation and safety evaluation of GM crops.}, }
@article {pmid39794549, year = {2025}, author = {Ahmed, R and Alghamdi, WN and Alharbi, FR and Alatawi, HD and Alenezi, KM and Alanazi, TF and Elsherbiny, NM}, title = {CRISPR/Cas9 System as a Promising Therapy in Thalassemia and Sickle Cell Disease: A Systematic Review of Clinical Trials.}, journal = {Molecular biotechnology}, volume = {}, number = {}, pages = {}, pmid = {39794549}, issn = {1559-0305}, abstract = {Clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is a new gene editing tool that represents a revolution in gene therapy. This study aimed to review the clinical trials conducted to evaluate the efficacy and safety of the CRISPR/Cas9 system in treating thalassemia and sickle cell disease (SCD). We searched relevant literature using "CRISPR Cas", "thalassemia", "sickle cell" and "clinical trial" as subject terms in PubMed, Cochrane, Web of Science, and Google Scholar up to December 3rd, 2023. Following the PIO format (Patients, Intervention, Outcome), PRISMA guidelines were followed in the study selection, data extraction, and quality assessment processes. Out of 110 publications, 6 studies met our eligibility criteria with a total of 115 patients involved. CRISPR/Cas9 system was used to disrupt BCL11A gene enhancer in 4 studies and to disrupt γ-globin gene promoters in 2 studies. Patients demonstrated significant activation of fetal hemoglobin, elevated total hemoglobin, transfusion independence in thalassemia, and repression of vaso-occlusive episodes in SCD. Using CRISPR/Cas9 system to directly disrupt genes provides a safe and potential one-time functional cure for thalassemia and SCD, suggesting CRISPR/Cas9 as a potential therapeutic tool for the treatment of inherited hematological disorders.}, }
@article {pmid39747689, year = {2025}, author = {Vaysset, H and Meers, C and Cury, J and Bernheim, A and Sternberg, SH}, title = {Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons.}, journal = {Nature microbiology}, volume = {10}, number = {1}, pages = {20-27}, pmid = {39747689}, issn = {2058-5276}, support = {2239685//National Science Foundation (NSF)/ ; GM143924//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; PECAN 101040529//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, mesh = {*DNA Transposable Elements/genetics ; *Evolution, Molecular ; *Transposases/genetics/metabolism ; *Phylogeny ; *Bacteria/genetics ; Archaea/genetics ; Eukaryota/genetics ; Archaeal Proteins/genetics/metabolism ; RNA, Small Nucleolar/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; RNA, Archaeal/genetics ; }, abstract = {Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS110-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms.}, }
@article {pmid39676659, year = {2025}, author = {Lee, JM and Zeng, J and Liu, P and Nguyen, MA and Suchenski Loustaunau, D and Bauer, DE and Kurt Yilmaz, N and Wolfe, SA and Schiffer, CA}, title = {Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, doi = {10.1093/nar/gkae1217}, pmid = {39676659}, issn = {1362-4962}, support = {R01 AI150478/AI/NIAID NIH HHS/United States ; DDF #2022092//Doris Duke Foundation/ ; R01AI150478/NH/NIH HHS/United States ; //University of Massachusetts Chan Medical School/ ; R01AI150478/NH/NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *Cytosine/chemistry/metabolism ; Humans ; *Ribonucleoproteins/metabolism/genetics ; *CRISPR-Cas Systems ; Animals ; Mice ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; }, abstract = {Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.}, }
@article {pmid39673524, year = {2025}, author = {Prajapat, MK and Maria, AG and Vidigal, JA}, title = {CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, doi = {10.1093/nar/gkae1138}, pmid = {39673524}, issn = {1362-4962}, support = {1ZIABC011810-02/NH/NIH HHS/United States ; /RC/CCR NIH HHS/United States ; 1ZIABC011810-02/NH/NIH HHS/United States ; /RC/CCR NIH HHS/United States ; }, mesh = {*MicroRNAs/genetics/metabolism ; Binding Sites/genetics ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; Cell Line ; Mutation ; HEK293 Cells ; }, abstract = {Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates confounding attempts at assigning function to the binding site. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.}, }
@article {pmid39657754, year = {2025}, author = {Kiernan, KA and Kwon, J and Merrill, BJ and Simonović, M}, title = {Structural basis of Cas9 DNA interrogation with a 5' truncated sgRNA.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, doi = {10.1093/nar/gkae1164}, pmid = {39657754}, issn = {1362-4962}, support = {R01 GM139894/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; F31 GM143822/GM/NIGMS NIH HHS/United States ; F31GM143822/GM/NIGMS NIH HHS/United States ; R01 GM097042/GM/NIGMS NIH HHS/United States ; 75N91019D00024/CA/NCI NIH HHS/United States ; F31GM143822/GM/NIGMS NIH HHS/United States ; 75N91019D00024/CA/NCI NIH HHS/United States ; }, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; Nucleic Acid Conformation ; Gene Editing/methods ; }, abstract = {The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent issue for using this system in cells. Studies have shown that the use of 5' truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15 nucleotides (nts) prevents full Cas9 activation without compromising on-target binding. Here, we use biochemistry and cryogenic electron microscopy to investigate Cas9 structure and activity when bound to a 14-nt sgRNA. Our structures reveal that the shortened path of the displaced non-target strand (NTS) sterically occludes docking of the HNH L1 linker and prevents proper positioning of the nuclease domains. We show that cleavage inhibition can be alleviated by either artificially melting the protospacer adjacent motif (PAM)-distal duplex or providing a supercoiled substrate. Even though Cas9 forms a stable complex with its target, we find that plasmid cleavage is ∼1000-fold slower with a 14-nt sgRNA than with a full-length 20-nt sgRNA. Our results provide a structural basis for Cas9 target binding with 5' truncated sgRNAs and underline the importance of PAM-distal NTS availability in promoting Cas9 activation.}, }
@article {pmid39793042, year = {2025}, author = {Ngo, W and Peukes, J and Baldwin, A and Xue, ZW and Hwang, S and Stickels, RR and Lin, Z and Satpathy, AT and Wells, JA and Schekman, R and Nogales, E and Doudna, JA}, title = {Mechanism-guided engineering of a minimal biological particle for genome editing.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {122}, number = {1}, pages = {e2413519121}, doi = {10.1073/pnas.2413519121}, pmid = {39793042}, issn = {1091-6490}, support = {1R21HL173710-01//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; DE-AC52-07NA27344//DOE | NNSA | Lawrence Livermore National Laboratory (LLNL)/ ; 1R01CA248323-01//HHS | National Institutes of Health (NIH)/ ; PDF-578176-2023//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; ALTF 1031-2021//European Molecular Biology Organization (EMBO)/ ; 1R01CA248323-01//HHS | National Institutes of Health (NIH)/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Ribonucleoproteins/metabolism/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; Genetic Vectors ; }, abstract = {The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.}, }
@article {pmid39792571, year = {2025}, author = {Islam, MS and Habib, MA and Tonu, NS and Haque, MS and Rahman, MM}, title = {Beyond Serology: A Meta-Analysis of Advancements in Molecular Detection of Brucella spp. in Seronegative Animals and Biological Samples.}, journal = {Veterinary medicine and science}, volume = {11}, number = {1}, pages = {e70200}, doi = {10.1002/vms3.70200}, pmid = {39792571}, issn = {2053-1095}, mesh = {Animals ; *Brucella/isolation & purification/immunology ; *Brucellosis/veterinary/diagnosis/microbiology ; Molecular Diagnostic Techniques/veterinary/methods ; }, abstract = {BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp., affecting various animals and humans, leading to significant economic and public health impacts. Traditional diagnostic methods, mainly serological, often fail to detect seronegative carriers, which continue to spread the infection.
OBJECTIVE: This review aims to highlight advancements in molecular diagnostics that address these limitations.
METHODS: A systematic search of PubMed, Web of Science and Scopus was conducted, focusing on studies using seronegative, PCR, qPCR and biosensor-based techniques. Data extraction and meta-analyses were performed, evaluating pooled detection rates and heterogeneity.
RESULTS: Through analysis of existing studies, we review key molecular techniques, including PCR, LAMP and biosensor-based assays, which offer high sensitivity and specificity by detecting bacterial DNA directly, thus overcoming the challenges of antibody-based tests. Meta-analysis of detection rates across different studies showed significant variability, with rates ranging from 0.96% to 100%, highlighting differences in sample types, animal species and regions. The pooled detection proportion from random-effects models was 35.08%, indicating that many seronegative animals still carry Brucella spp. A forest plot analysis further confirmed heterogeneity in detection, underlining the importance of using molecular diagnostics alongside serological tests to identify hidden carriers.
CONCLUSION: Innovations like nanoparticle-enhanced biosensors and CRISPR-Cas systems show promise for rapid, on-site diagnostics. The findings suggest that integrating molecular methods with traditional serology can improve surveillance and disease management. Future research should focus on developing portable, field-ready diagnostic devices and standardised protocols, along with exploring novel biomarkers to detect latent infections. A collaborative One Health approach, involving veterinary, public health and environmental sectors, is essential for comprehensive disease control and eradication efforts.}, }
@article {pmid39789283, year = {2025}, author = {Hassan, YM and Mohamed, AS and Hassan, YM and El-Sayed, WM}, title = {Recent developments and future directions in point-of-care next-generation CRISPR-based rapid diagnosis.}, journal = {Clinical and experimental medicine}, volume = {25}, number = {1}, pages = {33}, pmid = {39789283}, issn = {1591-9528}, mesh = {Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; Point-of-Care Systems ; SARS-CoV-2/genetics/isolation & purification ; Point-of-Care Testing ; Molecular Diagnostic Techniques/methods ; }, abstract = {The demand for sensitive, rapid, and affordable diagnostic techniques has surged, particularly following the COVID-19 pandemic, driving the development of CRISPR-based diagnostic tools that utilize Cas effector proteins (such as Cas9, Cas12, and Cas13) as viable alternatives to traditional nucleic acid-based detection methods. These CRISPR systems, often integrated with biosensing and amplification technologies, provide precise, rapid, and portable diagnostics, making on-site testing without the need for extensive infrastructure feasible, especially in underserved or rural areas. In contrast, traditional diagnostic methods, while still essential, are often limited by the need for costly equipment and skilled operators, restricting their accessibility. As a result, developing accessible, user-friendly solutions for at-home, field, and laboratory diagnostics has become a key focus in CRISPR diagnostic innovations. This review examines the current state of CRISPR-based diagnostics and their potential applications across a wide range of diseases, including cancers (e.g., colorectal and breast cancer), genetic disorders (e.g., sickle cell disease), and infectious diseases (e.g., tuberculosis, malaria, Zika virus, and human papillomavirus). Additionally, the integration of machine learning (ML) and artificial intelligence (AI) to enhance the accuracy, scalability, and efficiency of CRISPR diagnostics is discussed, alongside the challenges of incorporating CRISPR technologies into point-of-care settings. The review also explores the potential for these cutting-edge tools to revolutionize disease diagnosis and personalized treatment in the future, while identifying the challenges and future directions necessary to address existing gaps in CRISPR-based diagnostic research.}, }
@article {pmid39789078, year = {2025}, author = {Lee, D and Muir, P and Lundberg, S and Lundholm, A and Sandegren, L and Koskiniemi, S}, title = {A CRISPR-Cas9 system protecting E. coli against acquisition of antibiotic resistance genes.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {1545}, pmid = {39789078}, issn = {2045-2322}, mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics/drug effects ; Drug Resistance, Bacterial/genetics ; Gene Transfer, Horizontal ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; }, abstract = {Antimicrobial resistance (AMR) is an increasing problem worldwide, and new treatment options for bacterial infections are direly needed. Engineered probiotics show strong potential in treating or preventing bacterial infections. However, one concern with the use of live bacteria is the risk of the bacteria acquiring genes encoding for AMR or virulence factors through horizontal gene transfer (HGT), and the transformation of the probiotic into a superbug. Therefore, we developed an engineered CRISPR-Cas9 system that protects bacteria from horizontal gene transfer. We synthesized a CRISPR locus targeting eight AMR genes and cloned this with the Cas9 and transacting tracrRNA on a medium copy plasmid. We next evaluated the efficiency of the system to block HGT through transformation, transduction, and conjugation. Our results show that expression of the CRISPR-Cas9 system successfully protects E. coli MG1655 from acquiring the targeted resistance genes by transformation or transduction with 2-3 logs of protection depending on the system for transfer and the target gene. Furthermore, we show that the system blocks conjugation of a set of clinical plasmids, and that the system is also able to protect the probiotic bacterium E. coli Nissle 1917 from acquiring AMR genes.}, }
@article {pmid39789054, year = {2025}, author = {Li, W and Jiang, X and Wang, W and Hou, L and Cai, R and Li, Y and Gu, Q and Chen, Q and Ma, P and Tang, J and Guo, M and Chuai, G and Huang, X and Zhang, J and Liu, Q}, title = {Author Correction: Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {535}, doi = {10.1038/s41467-025-55913-y}, pmid = {39789054}, issn = {2041-1723}, }
@article {pmid39788669, year = {2025}, author = {Li, X and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y and Li, Y}, title = {Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343515}, doi = {10.1016/j.aca.2024.343515}, pmid = {39788669}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/chemistry/metabolism/antagonists & inhibitors ; Nucleic Acids/chemistry ; Bacterial Proteins ; }, abstract = {BACKGROUND: In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.
RESULTS: To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences.
SIGNIFICANCE AND NOVELTY: Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.}, }
@article {pmid39788657, year = {2025}, author = {Siler, T and Stanley, L and Saleem, M and Badalyan, A}, title = {A non-covalently bound redox indicator for electrochemical CRISPR-Cas12a and DNase I biosensors.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343480}, doi = {10.1016/j.aca.2024.343480}, pmid = {39788657}, issn = {1873-4324}, mesh = {*Biosensing Techniques/methods ; *Oxidation-Reduction ; *Electrochemical Techniques ; *DNA, Single-Stranded/chemistry/metabolism ; *Deoxyribonuclease I/metabolism/chemistry ; *CRISPR-Cas Systems ; Methylene Blue/chemistry ; CRISPR-Associated Proteins/metabolism/chemistry ; }, abstract = {A rapid and accurate biosensor for detecting disease biomarkers at point-of-care is essential for early disease diagnosis and preventing pandemics. CRISPR-Cas12a is a promising recognition element for DNA biosensors due to its programmability, specificity, and deoxyribonuclease activity initiated in the presence of a biomarker. The current electrochemical CRISPR-Cas12a-based biosensors utilize the single-stranded DNA (ssDNA) self-assembled on an electrode surface and covalently modified with the redox indicator, usually methylene blue (MB). In the presence of a biomarker, the nuclease domain is activated and cleaves ssDNA, decreasing the redox indicator signal. The covalent attachment of the MB to the ssDNA implies complexity and a higher production cost. Alternatively, some redox indicators can noncovalently bind to the ssDNA. Although such indicators have been applied for electrochemical nucleic acid detection, their potential for electrochemical CRISPR-Cas-based biosensors has not been explored. In this work, a ruthenium complex, [Ru(NH3)6][3+], was investigated as a redox indicator non-covalently binding to the ssDNA. Voltammetric studies and the optimization resulted in a simple and robust electrochemical method that was tested for deoxyribonuclease I (DNase I) activity detection and applied in the CRISPR-Cas12a-based biosensor for viral DNA (HPV-16). The biosensors revealed good analytical properties and represent an alternative to reported biosensors for nuclease activity requiring a covalent attachment of the redox indicator. Moreover, the developed method offers prospects for advancement and can be transformed to operate with other Cas nucleases to detect RNA and other analytes.}, }
@article {pmid39788645, year = {2025}, author = {Chen, Y and Zhao, R and Hu, X and Wang, X}, title = {The current status and future prospects of CRISPR-based detection of monkeypox virus: A review.}, journal = {Analytica chimica acta}, volume = {1336}, number = {}, pages = {343295}, doi = {10.1016/j.aca.2024.343295}, pmid = {39788645}, issn = {1873-4324}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Monkeypox virus/genetics ; Mpox (monkeypox)/diagnosis ; }, abstract = {BACKGROUND: The current pandemic of 2022 global mpox (formerly known as monkeypox), caused by infection with monkeypox virus (MPXV), has now reached over 120 countries. This constitutes a critical public health issue requiring effective disease management and surveillance. Rapid and reliable diagnosis is conducive to the control of infection, early intervention, and timely treatment. Clinical laboratories use various conventional diagnostic methods for detecting MPXV, including PCR, which can be regarded as a gold-standard diagnostic method. However, the application of PCR is limited by its requirements for high-cost equipment, skilled professionals, and a laboratory setting.
RESULTS: Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems have provided promising prospects for the rapid, sensitive, and specific detection of infectious diseases, especially in point-of-care settings. Over the past 2 years, an increasing number of researchers have concentrated on the application of the CRISPR method to mpox diagnosis. In the majority of cases, a two-step method was chosen, with CRISPR/Cas12a and recombinase polymerase amplification (RPA) as pre-amplification methods, followed by a fluorescence readout. Different strategies have been applied to overcome the encountered limitations of CRISPR detection, but no consensus on an integrated solution has been achieved. Thus, the application of the CRISPR/Cas system in mpox detection requires further exploration and improvement.
SIGNIFICANCE: This review discusses contemporary studies on MPXV CRISPR detection systems and the strategies proposed to address the challenges faced by CRISPR diagnosis with the hope of helping the development of CRISPR detection methods and improving pathogen detection technologies.}, }
@article {pmid39788509, year = {2024}, author = {Wu, K and Xie, J and Liu, X and Yang, D and Wang, Y and Zhao, W and Shang, X and Jiang, L}, title = {Construction and biological function of Toxoplasma gondii rop41 gene knockout strain.}, journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences}, volume = {49}, number = {8}, pages = {1200-1209}, doi = {10.11817/j.issn.1672-7347.2024.240179}, pmid = {39788509}, issn = {1672-7347}, support = {32170510//the National Natural Science Foundation of China/ ; 20240026020055//the Innovation Training Program of Central South University/ ; }, mesh = {*Toxoplasma/genetics ; *Protozoan Proteins/genetics/metabolism ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Plasmids/genetics ; Virulence Factors/genetics ; Animals ; }, abstract = {OBJECTIVES: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable T. gondii to invade host cells. This study aims to construct a T. gondii rhoptry protein 41 (rop41/ROP41) gene knockout strain and preliminarily investigate the biological function of rop41.
METHODS: Using CRISPR/Cas9 technology, a specific single-guide RNA (sgRNA) for the target gene was designed and linked to a recombinant plasmid. Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes. The recombinant plasmid and the homologous fragments were electroporated into T. gondii, and PCR identification was performed after drug selection and monoclonal screening. Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T. gondii in host cells. Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity. The STRING database was utilized to construct a protein-protein interaction (PPI) network, and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.
RESULTS: The T. gondiirop41 gene knockout strain (RH Δku80Δrop41) was successfully constructed and stably inherited. Plaque assays showed that compared with the parental strain, the number of plaques formed by the rop41 gene knockout strain did not significantly decrease, but the reduction in plaque size was statistically significant (P<0.05). After the rop41 gene was knocked out, the invasion ability of T. gondii was reduced, but there was no statistically significant difference in its proliferation ability (P>0.05). The PPI network revealed that ROP41 was associated with other protein kinases and autophagy-related proteins. Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction, biosynthesis, metabolism, and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.
CONCLUSIONS: The T. gondii RH Δku80Δrop41 strain has been successfully constructed. ROP41 primarily affects the ability of T. gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T. gondii and the host.}, }
@article {pmid39788098, year = {2025}, author = {Lin, J and Bhoobalan-Chitty, Y and Peng, X}, title = {Cad1 turns ATP into phage poison.}, journal = {Cell host & microbe}, volume = {33}, number = {1}, pages = {8-10}, doi = {10.1016/j.chom.2024.12.013}, pmid = {39788098}, issn = {1934-6069}, mesh = {*Adenosine Triphosphate/metabolism ; *Bacteriophages/genetics/physiology/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics/virology/metabolism ; }, abstract = {Type III CRISPR-Cas executes a multifaceted anti-phage response, activating effectors such as a nuclease or membrane depolarizer. In a recent Cell paper, Baca and Majumder et al.[1] report an accessory effector, Cad1, which deaminates ATP into ITP, causing ITP accumulation and host growth arrest, thereby inhibiting phage propagation.}, }
@article {pmid39784310, year = {2025}, author = {Dai, J and Wu, B and Ai, F and Yang, Z and Lu, Y and Zinian, C and Zeng, K and Zhang, Z}, title = {Exploiting the Potential of Spherical PAM Antenna for Enhanced CRISPR-Cas12a: A Paradigm Shift toward a Universal Amplification-Free Nucleic Acid Test Platform.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c04871}, pmid = {39784310}, issn = {1520-6882}, abstract = {The CRISPR-Cas12a system has shown tremendous potential for developing efficient biosensors. Albeit important, current CRISPR-Cas system-based diagnostic technologies (CRISPR-DX) highly rely on an additional preamplification procedure to obtain high sensitivity, inevitably leading to issues such as complicated assay workflow, cross-contamination, etc. Herein, a spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a system is fabricated for universal amplification-free nucleic acid detection with a detection limit of subfemtomolar. Meanwhile, the clinical detection capability of this sensor was further verified using gold-standard real-time quantitative polymerase chain reaction through Mycobacterium tuberculosis measurement, which demonstrated its good reliability for practical applications. Importantly, its excellent sensitivity is mainly ascribed to high efficiency of target search induced by a localized PAM-enriched microenvironment and improved catalytic activity of Cas12a (up to 4 folds). Our strategy provides some new insights for rapid and sensitive detection of nucleic acids in an amplification-free fashion.}, }
@article {pmid39781718, year = {2025}, author = {Saberi, F and Yousefi-Najafabadi, Z and Shams, F and Dehghan, Z and Ahmadi, S and Pilehchi, T and Noori, E and Esmaeelzadeh, Z and Bazgiri, M and Mohammadi, R and Khani, F and Sameni, M and Moradbeigi, P and Kardar, GA and Salehi, M and Teng, Y and Jajarmi, V}, title = {CRISPR/Cas System: A Powerful Strategy to Improve Monogenic Human Diseases as Therapeutic Delivery; Current Applications and Challenges.}, journal = {Current gene therapy}, volume = {}, number = {}, pages = {}, doi = {10.2174/0115665232345516241119070150}, pmid = {39781718}, issn = {1875-5631}, abstract = {The 5,000 to 8,000 monogenic diseases are inherited disorders leading to mutations in a single gene. These diseases usually appear in childhood and sometimes lead to morbidity or premature death. Although treatments for such diseases exist, gene therapy is considered an effective and targeted method and has been used in clinics for monogenic diseases since 1989. Monogenic diseases are good candidates for novel therapeutic technologies like gene editing approaches to repair gene mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, the pioneer and effective gene editing tool, are utilized for ex vivo and in vivo treatment of monogenic diseases. The current review provides an overview of recent therapeutic applications of CRISPR-based gene editing in monogenic diseases in in vivo and ex vivo models. Furthermore, this review consolidates strategies aimed at providing new treatment options with gene therapy, thereby serving as a valuable reference for advancing the treatment landscape for patients with monogenic disorders.}, }
@article {pmid39779704, year = {2025}, author = {Kempthorne, L and Vaizoglu, D and Cammack, AJ and Carcolé, M and Roberts, MJ and Mikheenko, A and Fisher, A and Suklai, P and Muralidharan, B and Kroll, F and Moens, TG and Yshii, L and Verschoren, S and Hölbling, BV and Moreira, FC and Katona, E and Coneys, R and de Oliveira, P and Zhang, YJ and Jansen, K and Daughrity, LM and McGown, A and Ramesh, TM and Van Den Bosch, L and Lignani, G and Rahim, AA and Coyne, AN and Petrucelli, L and Rihel, J and Isaacs, AM}, title = {Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {459}, pmid = {39779704}, issn = {2041-1723}, support = {648716 - C9ND//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 217150/Z/19/Z//Wellcome Trust (Wellcome)/ ; }, mesh = {*C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/therapy ; Humans ; *Frontotemporal Dementia/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *RNA, Antisense/genetics ; Mice ; HEK293 Cells ; *Induced Pluripotent Stem Cells/metabolism ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Neurons/metabolism ; Genetic Therapy/methods ; }, abstract = {The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.}, }
@article {pmid39779681, year = {2025}, author = {McCallister, TX and Lim, CKW and Singh, M and Zhang, S and Ahsan, NS and Terpstra, WM and Xiong, AY and Zeballos C, MA and Powell, JE and Drnevich, J and Kang, Y and Gaj, T}, title = {A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {460}, pmid = {39779681}, issn = {2041-1723}, support = {1U01NS122102-01A1//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556-01A1//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5R01GM141296//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; 20-IIP-516//Amyotrophic Lateral Sclerosis Association (ALS Association)/ ; }, mesh = {*Amyotrophic Lateral Sclerosis/genetics/pathology/metabolism ; *C9orf72 Protein/genetics/metabolism ; *Frontotemporal Dementia/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Humans ; Animals ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Motor Neurons/metabolism/pathology ; Mice ; }, abstract = {An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.}, }
@article {pmid39779650, year = {2025}, author = {Aydin, A and Yerlikaya, BA and Yerlikaya, S and Yilmaz, NN and Kavas, M}, title = {CRISPR-mediated mutation of cytokinin signaling genes (SlHP2 and SlHP3) in tomato: Morphological, physiological, and molecular characterization.}, journal = {The plant genome}, volume = {18}, number = {1}, pages = {e20542}, pmid = {39779650}, issn = {1940-3372}, support = {PYO.ZRT.1904.22.006//Ondokuz Mayis Üniversitesi/ ; }, mesh = {*Solanum lycopersicum/genetics/physiology ; *Cytokinins/metabolism ; *Signal Transduction ; Mutation ; CRISPR-Cas Systems ; Droughts ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; Gene Editing ; }, abstract = {Synergistic and antagonistic relationships between cytokinins and other plant growth regulators are important in response to changing environmental conditions. Our study aimed to determine the functions of SlHP2 and SlHP3, two members of cytokinin signaling in tomato, in drought stress response using CRISPR/Cas9-mediated mutagenesis. Ten distinct genome-edited lines were generated via Agrobacterium tumefaciens-mediated gene transfer and confirmed through Sanger sequencing. Stress experiments were conducted with two of these lines (slhp2,3-10 and slhp2,3-11), which harbored homozygous mutations in both genes. The responses of two lines carrying homozygous mutations in both genes under polyethylene glycol (PEG)-induced stress were examined using morphological, physiological, biochemical, and molecular methods. The genome-edited lines demonstrated enhanced water retention, reduced stomatal density, and less oxidative damage compared to the wild-type plants under PEG-induced stress. Moreover, the slhp2,3 double mutant plants exhibited improved root growth, showcasing their superior drought tolerance over wild-type plants by accessing deeper water sources and maintaining hydration in water-limited environments. To investigate the involvement of cytokinin signaling regulators and genes associated with stomatal formation and differentiation, the expression of genes (Speechless [SPCH], FAMA, MUTE, TMM, HB25, HB31, RR6, RR7, and Solyc02g080860) was assessed. The results revealed that all regulators were downregulated, with SPCH, TMM, RR7, and RR6 showing significant reductions under PEG-induced stress. These results emphasize the promise of utilizing CRISPR/Cas9 to target cytokinin signaling pathways, enhancing drought tolerance in tomatoes through improvements in water retention and root growth, along with a reduction in stomatal density and malondialdehyde content.}, }
@article {pmid39779219, year = {2025}, author = {Tang, JX and Cabrera-Orefice, A and Meisterknecht, J and Taylor, LS and Monteuuis, G and Stensland, ME and Szczepanek, A and Stals, K and Davison, J and He, L and Hopton, S and Nyman, TA and Jackson, CB and Pyle, A and Winter, M and Wittig, I and Taylor, RW}, title = {COA5 has an essential role in the early stage of mitochondrial complex IV assembly.}, journal = {Life science alliance}, volume = {8}, number = {3}, pages = {}, pmid = {39779219}, issn = {2575-1077}, mesh = {Humans ; Male ; *Mitochondria/metabolism/genetics ; *Electron Transport Complex IV/metabolism/genetics ; Female ; Mitochondrial Proteins/metabolism/genetics ; Mitochondrial Diseases/genetics/metabolism/pathology ; Pedigree ; CRISPR-Cas Systems ; Fibroblasts/metabolism ; Mutation, Missense ; }, abstract = {Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.}, }
@article {pmid39777467, year = {2025}, author = {Xu, X and Lv, X and Liu, Y and Li, J and Du, G and Chen, J and Ledesma-Amaro, R and Liu, L}, title = {CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis.}, journal = {Nucleic acids research}, volume = {53}, number = {1}, pages = {}, pmid = {39777467}, issn = {1362-4962}, support = {32200050//National Natural Science Foundation of China/ ; BK20221079//Natural Science Foundation of Jiangsu Province/ ; JUSRP52019A//Fundamental Research Funds for the Central Universities/ ; 2020YFA0908300//National Key Research and Development Program of China/ ; BK20233003//Jiangsu Basic Research Center for Synthetic Biology/ ; }, mesh = {*CRISPR-Cas Systems ; *Bacillus subtilis/genetics/metabolism ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA Stability ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; }, abstract = {Developing efficient gene regulation tools is essential for optimizing microbial cell factories, but most existing tools only modulate gene expression at the transcriptional level. Regulation at the translational level provides a faster dynamic response, whereas developing a programmable, efficient and multiplexed translational regulation tool remains a challenge. Here, we have developed CRISPRi and CRISPRa systems based on hfCas13X that can regulate gene translation in Bacillus subtilis. First, we constructed a CRISPRi system to regulate gene translation based on catalytically deactivated hfCas13X (dhfCas13X). Second, we designed unique mRNA-crRNA pairs to construct DiCRISPRa (degradation-inhibited CRISPRa) and TsCRISPRa (translation-started CRISPRa) systems, which can activate downstream gene translation by enhancing mRNA stability or initiating mRNA translation. In addition, we found that fusing dhfCas13X with the RNA-binding chaperone BHfq significantly improved the activation efficiency of the DiCRISPRa and TsCRISPRa systems (43.2-fold). Finally, we demonstrated that the constructed CRISPR systems could be used to optimize the metabolic networks of two biotechnologically relevant compounds, riboflavin and 2'-fucosyllactose, increasing their titers by 3- and 1.2-fold, respectively. The CRISPRa and CRISPRi systems developed here provide new tools for the regulation of gene expression at the translation level and offer new ideas for the construction of CRISPRa systems.}, }
@article {pmid39775780, year = {2025}, author = {Tong, G and Nath, P and Hiruta, Y and Citterio, D}, title = {Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device.}, journal = {Lab on a chip}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4lc01039f}, pmid = {39775780}, issn = {1473-0189}, abstract = {Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM - among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.}, }
@article {pmid39775585, year = {2024}, author = {Zhang, WW and Matlashewski, G}, title = {Evidence for gene essentiality in Leishmania using CRISPR.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0316331}, pmid = {39775585}, issn = {1932-6203}, mesh = {*Genes, Essential ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Leishmania/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Protozoan ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Ribosomal/genetics ; Plasmids/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; }, abstract = {The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.}, }
@article {pmid39774105, year = {2025}, author = {Ocampo, RF and Bravo, JPK and Dangerfield, TL and Nocedal, I and Jirde, SA and Alexander, LM and Thomas, NC and Das, A and Nielson, S and Johnson, KA and Brown, CT and Butterfield, CN and Goltsman, DSA and Taylor, DW}, title = {DNA targeting by compact Cas9d and its resurrected ancestor.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {457}, pmid = {39774105}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; HEK293 Cells ; Endonucleases/metabolism/genetics/chemistry ; Protein Domains ; }, abstract = {Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.}, }
@article {pmid39774003, year = {2025}, author = {Claiborne, DT and Detwiler, Z and Docken, SS and Borland, TD and Cromer, D and Simkhovich, A and Ophinni, Y and Okawa, K and Bateson, T and Chen, T and Hudson, W and Trifonova, R and Davenport, MP and Ho, TW and Boutwell, CL and Allen, TM}, title = {High frequency CCR5 editing in human hematopoietic stem progenitor cells protects xenograft mice from HIV infection.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {446}, pmid = {39774003}, issn = {2041-1723}, support = {U19 HL156247/HL/NHLBI NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; }, mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; Animals ; *Hematopoietic Stem Cells/metabolism/virology ; *HIV Infections/virology/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Mice ; Heterografts ; HIV-1/genetics ; Mice, SCID ; }, abstract = {The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.}, }
@article {pmid39773393, year = {2025}, author = {Bogut, A and Kołodziejek, A and Minnich, SA and Hovde, CJ}, title = {CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli.}, journal = {Archivum immunologiae et therapiae experimentalis}, volume = {73}, number = {1}, pages = {}, pmid = {39773393}, issn = {1661-4917}, mesh = {Humans ; *Enterohemorrhagic Escherichia coli/genetics ; *CRISPR-Cas Systems ; Animals ; *Escherichia coli Infections/diagnosis/therapy/immunology ; *Gene Editing/methods ; Cattle ; Virulence/genetics ; Hemolytic-Uremic Syndrome/diagnosis/therapy/immunology/microbiology ; Probiotics/therapeutic use ; Bacteriophages/genetics ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Following its discovery as an adaptive immune system in prokaryotes, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been developed into a multifaceted genome editing tool. This review compiles findings aimed at implementation of this technology for selective elimination or attenuation of enterohemorrhagic Escherichia coli (EHEC). EHEC are important zoonotic foodborne pathogens that cause hemorrhagic colitis and can progress to the life-threatening hemolytic uremic syndrome (HUS). Advancements in the application of CRISPR methodology include laboratory detection and identification of EHEC, genotyping, screening for pathogenic potential, and engineering probiotics to reduce microbial shedding by cattle, the primary source of human infection. Genetically engineered phages or conjugative plasmids have been designed to target and inactivate genes whose products are critical for EHEC virulence.}, }
@article {pmid39773308, year = {2025}, author = {Chen, H and Fang, HQ and Liu, JT and Chang, SY and Cheng, LB and Sun, MX and Feng, JR and Liu, ZM and Zhang, YH and Rosen, CJ and Liu, P}, title = {Atlas of Fshr expression from novel reporter mice.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39773308}, issn = {2050-084X}, mesh = {Animals ; *Receptors, FSH/genetics/metabolism ; Mice ; *Genes, Reporter ; Male ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; }, abstract = {The FSH-FSHR pathway has been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to several limitations. To address this challenge, we developed Fshr-ZsGreen reporter mice under the control of Fshr endogenous promoter using CRISPR-Cas9. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses,to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.}, }
@article {pmid39770835, year = {2024}, author = {Tuli, SR and Ali, MF and Jamal, TB and Khan, MAS and Fatima, N and Ahmed, I and Khatun, M and Sharmin, SA}, title = {Characterization and Molecular Insights of a Chromium-Reducing Bacterium Bacillus tropicus.}, journal = {Microorganisms}, volume = {12}, number = {12}, pages = {}, pmid = {39770835}, issn = {2076-2607}, abstract = {Environmental pollution from metal toxicity is a widespread concern. Certain bacteria hold promise for bioremediation via the conversion of toxic chromium compounds into less harmful forms, promoting environmental cleanup. In this study, we report the isolation and detailed characterization of a highly chromium-tolerant bacterium, Bacillus tropicus CRB14. The isolate is capable of growing on 5000 mg/L Cr (VI) in an LB (Luria Bertani) agar plate while on 900 mg/L Cr (VI) in LB broth. It shows an 86.57% reduction ability in 96 h of culture. It can also tolerate high levels of As, Cd, Co, Fe, Zn, and Pb. The isolate also shows plant growth-promoting potential as demonstrated by a significant activity of nitrogen fixation, phosphate solubilization, IAA (indole acetic acid), and siderophore production. Whole-genome sequencing revealed that the isolate lacks Cr resistance genes in their plasmids and are located on its chromosome. The presence of the chrA gene points towards Cr(VI) transport, while the absence of ycnD suggests alternative reduction pathways. The genome harbors features like genomic islands and CRISPR-Cas systems, potentially aiding adaptation and defense. Analysis suggests robust metabolic pathways, potentially involved in Cr detoxification. Notably, genes for siderophore and NRP-metallophore production were identified. Whole-genome sequencing data also provides the basis for molecular validation of various genes. Findings from this study highlight the potential application of Bacillus tropicus CRB14 for bioremediation while plant growth promotion can be utilized as an added benefit.}, }
@article {pmid39770798, year = {2024}, author = {Muriuki, R and Ndichu, M and Githigia, S and Svitek, N}, title = {CRISPR-Cas-Based Pen-Side Diagnostic Tests for Anaplasma marginale and Babesia bigemina.}, journal = {Microorganisms}, volume = {12}, number = {12}, pages = {}, pmid = {39770798}, issn = {2076-2607}, support = {7200AA20CA00022//USAID/ ; }, abstract = {Anaplasma marginale and Babesia bigemina are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of Babesia bigemina and Anaplasma marginale in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting Theileria parva infections in cattle, we have created two additional CRISPR-Cas12a assays targeting Anaplasma marginale and Babesia bigemina. The assays target the major surface protein 5 (MSP5) for A. marginale and rhoptry-associated protein 1a (RAP1a) for B. bigemina. These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10[2] DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose A. marginale and B. bigemina infections.}, }
@article {pmid39770347, year = {2024}, author = {de Sousa, RMP and Garcia, LS and Lemos, FS and de Campos, VS and Machado Ferreira, E and de Almeida, NAA and Maron-Gutierrez, T and de Souza, EM and de Paula, VS}, title = {CRISPR/Cas9 Eye Drop HSV-1 Treatment Reduces Brain Viral Load: A Novel Application to Prevent Neuronal Damage.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, pmid = {39770347}, issn = {2076-0817}, support = {GM-GD 2022//National Council for Scientific and Technological Development/ ; 001//Coordenação de Aperfeicoamento de Pessoal de Nível Superior/ ; VPGDI 024 INT 22//Fundação Oswaldo Cruz/ ; 001//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; }, mesh = {Animals ; *Herpesvirus 1, Human/drug effects/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems/genetics ; Mice ; *Viral Load/drug effects ; *Mice, Inbred BALB C ; *Antiviral Agents/pharmacology ; *Brain/virology/pathology/drug effects/metabolism ; *Herpes Simplex/drug therapy/virology ; Female ; Virus Replication/drug effects ; Disease Models, Animal ; Gene Editing/methods ; }, abstract = {Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.}, }
@article {pmid39770308, year = {2024}, author = {Islam, MM and Jung, DE and Shin, WS and Oh, MH}, title = {Colistin Resistance Mechanism and Management Strategies of Colistin-Resistant Acinetobacter baumannii Infections.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, doi = {10.3390/pathogens13121049}, pmid = {39770308}, issn = {2076-0817}, support = {Grant Nos. 2022R1F1A1071415 and NRF-RS-2023-00275307 and Grant No. 2019R1A6C1010033//National Research Foundation of Korea (NRF) funded by the Ministry of Education & Basic Science Research Capacity Enhancement Project through Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry o/ ; }, mesh = {*Acinetobacter baumannii/drug effects/genetics ; *Colistin/pharmacology/therapeutic use ; Humans ; *Acinetobacter Infections/drug therapy ; *Anti-Bacterial Agents/therapeutic use/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics/drug effects ; Drug Resistance, Bacterial/genetics/drug effects ; Animals ; }, abstract = {The emergence of antibiotic-resistant Acinetobacter baumannii (A. baumannii) is a pressing threat in clinical settings. Colistin is currently a widely used treatment for multidrug-resistant A. baumannii, serving as the last line of defense. However, reports of colistin-resistant strains of A. baumannii have emerged, underscoring the urgent need to develop alternative medications to combat these serious pathogens. To resist colistin, A. baumannii has developed several mechanisms. These include the loss of outer membrane lipopolysaccharides (LPSs) due to mutation of LPS biosynthetic genes, modification of lipid A (a constituent of LPSs) structure through the addition of phosphoethanolamine (PEtN) moieties to the lipid A component by overexpression of chromosomal pmrCAB operon genes and eptA gene, or acquisition of plasmid-encoded mcr genes through horizontal gene transfer. Other resistance mechanisms involve alterations of outer membrane permeability through porins, the expulsion of colistin by efflux pumps, and heteroresistance. In response to the rising threat of colistin-resistant A. baumannii, researchers have developed various treatment strategies, including antibiotic combination therapy, adjuvants to potentiate antibiotic activity, repurposing existing drugs, antimicrobial peptides, nanotechnology, photodynamic therapy, CRISPR/Cas, and phage therapy. While many of these strategies have shown promise in vitro and in vivo, further clinical trials are necessary to ensure their efficacy and widen their clinical applications. Ongoing research is essential for identifying the most effective therapeutic strategies to manage colistin-resistant A. baumannii. This review explores the genetic mechanisms underlying colistin resistance and assesses potential treatment options for this challenging pathogen.}, }
@article {pmid39770154, year = {2024}, author = {Shang, Z and Liu, S and Liu, D and Pei, X and Li, S and He, Y and Tong, Y and Liu, G}, title = {CRISPR/Cas12a with Universal crRNA for Indiscriminate Virus Detection.}, journal = {Molecules (Basel, Switzerland)}, volume = {29}, number = {24}, pages = {}, pmid = {39770154}, issn = {1420-3049}, support = {No. 22004005//National Natural Science Foundation of China/ ; 223777118D//Special Project on Biomedical Innovation/ ; }, mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/genetics/isolation & purification ; Humans ; *RNA, Viral/genetics/analysis ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics ; Biosensing Techniques/methods ; }, abstract = {Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to SARS-CoV-2, human papillomaviruses (HPV), HCOV-NL63, HCOV-HKU1, and miRNA biomarkers. Taking SARS-CoV-2 and HPV16 pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications.}, }
@article {pmid39769261, year = {2024}, author = {Taki, T and Morimoto, K and Mizuno, S and Kuno, A}, title = {KOnezumi-AID: Automation Software for Efficient Multiplex Gene Knockout Using Target-AID.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769261}, issn = {1422-0067}, support = {JP24ama121047//Japan Agency for Medical Research and Development/ ; JPMJFR221H//Japan Science and Technology Agency/ ; 24K18045//Japan Society for the Promotion of Science/ ; }, mesh = {*Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Humans ; *Software ; }, abstract = {With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.}, }
@article {pmid39769183, year = {2024}, author = {Kappler, M and Thielemann, L and Glaß, M and Caggegi, L and Güttler, A and Pyko, J and Blauschmidt, S and Gutschner, T and Taubert, H and Otto, S and Eckert, AW and Tavassol, F and Bache, M and Vordermark, D and Kaune, T and Rot, S}, title = {Functional and Biological Characterization of the LGR5Δ5 Splice Variant in HEK293T Cells.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769183}, issn = {1422-0067}, support = {NA//Dr. Kleist Stiftung (Foundation)/ ; NA//Open Access Publication Fund of the Martin Luther University Halle-Wittenberg/ ; }, mesh = {Humans ; *Receptors, G-Protein-Coupled/genetics/metabolism ; HEK293 Cells ; *Thrombospondins/genetics/metabolism ; Wnt Signaling Pathway/genetics ; Protein Isoforms/genetics/metabolism ; CRISPR-Cas Systems ; Cell Movement/genetics ; }, abstract = {The regulator of the canonical Wnt pathway, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is expressed in the stem cell compartment of several tissues and overexpressed in different human carcinomas. The isoform of the stem cell marker LGR5, named LGR5Δ5 and first described by our group, is associated with prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS). In a proof-of-principle analysis, the function of LGR5Δ5 was investigated in HEK293T cells, a model cell line of the Wnt pathway, compared to full-length LGR5 (FL) expression. The CRISPR/CAS knockout of LGR5 and LGR4 (thereby avoiding the side effects of LGR4) resulted in a loss of Wnt activity that cannot be restored by LGR5Δ5 but by LGR5FL rescue. The ability to migrate was not affected by LGR5Δ5, but was reduced by LGR5FL overexpression. The CRISPR/CAS of LGR4 and 5 induced radiosensitization, which was enhanced by the overexpression of LGR5FL or LGR5Δ5. RNA sequencing analysis revealed a significant increase in the ligand R-spondin 1 (RSPO1) level by LGR5Δ5. Furthermore, LGR5Δ5 appears to be involved in the regulation of genes related to the cytoskeleton, extracellular matrix stiffness, and angiogenesis, while LGR5FL is associated with the regulation of collagens and histone proteins.}, }
@article {pmid39769084, year = {2024}, author = {Bairqdar, A and Karitskaya, PE and Stepanov, GA}, title = {Expanding Horizons of CRISPR/Cas Technology: Clinical Advancements, Therapeutic Applications, and Challenges in Gene Therapy.}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769084}, issn = {1422-0067}, support = {075-15-2021-1085//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Gene Editing/methods ; Clinical Trials as Topic ; Animals ; }, abstract = {CRISPR-Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR-Cas-based therapies. This review examines the current landscape of CRISPR-Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial.}, }
@article {pmid39769020, year = {2024}, author = {Sun, X and Fu, Q and Song, Y and Deng, X and Li, Y and Wu, K and Li, S and Fu, J}, title = {Research Progress and Prospects of Molecular Breeding in Bermudagrass (Cynodon dactylon).}, journal = {International journal of molecular sciences}, volume = {25}, number = {24}, pages = {}, pmid = {39769020}, issn = {1422-0067}, support = {2024LZGC00303//Key R & D Plan of Shandong Province/ ; }, mesh = {*Cynodon/genetics ; *Plant Breeding/methods ; Genomics/methods ; Gene Editing/methods ; Stress, Physiological ; Genome, Plant ; }, abstract = {Bermudagrass (Cynodon dactylon L.) is a warm-season grass species of significant ecological and economic importance. It is widely utilized in turf management and forage production due to its resilience to drought, salt, and other environmental stresses. Recent advancements in molecular breeding, particularly through genomics technology and gene editing, have enabled the efficient identification of key genes associated with stress tolerance and turf quality. The use of techniques such as overexpression and CRISPR/Cas has enhanced resistance to drought, salt, cold, and heat, while the application of molecular markers has accelerated the development of superior varieties. The integration of multi-omics, such as genomics, transcriptomics, and proteomics, provides deeper insights into the molecular mechanisms of bermudagrass, thereby improving breeding efficiency and precision. Additionally, artificial intelligence is emerging as a powerful tool for analyzing genomic data, predicting optimal trait combinations, and accelerating breeding processes. These technologies, when combined with traditional breeding methods, hold great potential for optimizing bermudagrass varieties for both turf and forage use. Future research will focus on further integrating these tools to address the challenges of breeding posed by climate change to breeding climate-resilient turf and forage crops.}, }
@article {pmid39768143, year = {2024}, author = {Zhang, XH and Tang, FL and Trouten, AM and Morad, M}, title = {Attempts to Create Transgenic Mice Carrying the Q3924E Mutation in RyR2 Ca[2+] Binding Site.}, journal = {Cells}, volume = {13}, number = {24}, pages = {}, pmid = {39768143}, issn = {2073-4409}, support = {R01HL153504/GF/NIH HHS/United States ; }, mesh = {Animals ; *Ryanodine Receptor Calcium Release Channel/genetics/metabolism ; *Mice, Transgenic ; Mice ; *Calcium/metabolism ; *Myocytes, Cardiac/metabolism ; Binding Sites ; Humans ; Mutation/genetics ; Female ; Calcium Signaling/genetics ; CRISPR-Cas Systems/genetics ; Caffeine/pharmacology ; }, abstract = {Over 200 point mutations in the ryanodine receptor (RyR2) of the cardiac sarcoplasmic reticulum (SR) are known to be associated with cardiac arrhythmia. We have already reported on the calcium signaling phenotype of a point mutation in RyR2 Ca[2+] binding site Q3925E expressed in human stem-cell-derived cardiomyocytes (hiPSC-CMs) that was found to be lethal in a 9-year-old girl. CRISPR/Cas9-gene-edited mutant cardiomyocytes carrying the RyR2-Q3925E mutation exhibited a loss of calcium-induced calcium release (CICR) and caffeine-triggered calcium release but continued to beat arrhythmically without generating significant SR Ca[2+] release, consistent with a remodeling of the calcium signaling pathway. An RNAseq heat map confirmed significant changes in calcium-associated genes, supporting the possibility of remodeling. To determine the in situ cardiac phenotype in an animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/- using the CRISPR/Cas9 technique. We obtained three homozygous and one chimera mice, but they all died before reaching 3 weeks of age, preventing the establishment of germline mutation transmission in their offspring. A histo-pathological analysis of the heart showed significant cardiac hypertrophy, suggesting the Q3924E-RyR2 mutation was lethal to the mice.}, }
@article {pmid39766827, year = {2024}, author = {Yang, R and Guo, H and Sun, J and Gui, T and Li, X and Qian, H and Chen, A}, title = {The ebony Gene in Silkworm Black Pupae Significantly Affects 30 K Proteins During the Pupal Stage.}, journal = {Genes}, volume = {15}, number = {12}, pages = {}, pmid = {39766827}, issn = {2073-4425}, support = {CARS-18-ZJ0101//China Agriculture Research System of MOF and MARA/ ; BE2020418//Key R & D plan of Jiangsu Province (Modern Agriculture)/ ; 2023-JC-YB-188//Basic Research Programs of the Shaanxi Provincial Science and Technology Department/ ; 2023-YBNY-134//Key Industrial Chain Projects of Shaanxi Provincial Government/ ; }, mesh = {Animals ; *Bombyx/genetics/growth & development/metabolism ; *Pupa/genetics/growth & development/metabolism ; *Insect Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Pigmentation/genetics ; CRISPR-Cas Systems ; }, abstract = {Background/Objectives: The body color and patterns of insects play important roles in foraging, evading predators, mating, thermoregulation, and environmental adaptation. During the rearing of the QiufengN silkworm strain, a mutant with black pupal cuticle (QiufengNBP) was discovered. Preliminary map-based cloning and sequence analysis indicated that the ebony gene might significantly influence the formation of the black pupa mutant and the expression of 30K proteins. This study aims to determine the function of the ebony gene and its effect on the expression of the 30K protein during the pupal stage; Methods and Results: We employed CRISPR/Cas9 gene-editing technology to knock out the ebony gene in the Nistari strain, resulting in individuals with black pupae, named Nistari Black Pupa (NisBP). This confirmed that the ebony gene plays a crucial role in black pupa formation. Two-dimensional electrophoresis (2-DE) analysis of the pupal cuticle of NisBP and its wild-type Nistari found that the ebony gene has a significant impact on the expression of 30K proteins, which are vital for embryonic development and serve as key storage proteins; Conclusions: This study is the first to demonstrate that the ebony gene affects the expression of 30K proteins, laying the foundation for further research on their functions and providing insights into the developmental mechanisms of silkworms.}, }
@article {pmid39766530, year = {2024}, author = {de la Fuente Tagarro, C and Martín-González, D and De Lucas, A and Bordel, S and Santos-Beneit, F}, title = {Current Knowledge on CRISPR Strategies Against Antimicrobial-Resistant Bacteria.}, journal = {Antibiotics (Basel, Switzerland)}, volume = {13}, number = {12}, pages = {}, pmid = {39766530}, issn = {2079-6382}, abstract = {CRISPR/Cas systems have emerged as valuable tools to approach the problem of antimicrobial resistance by either sensitizing or lysing resistant bacteria or by aiding in antibiotic development, with successful applications across diverse organisms, including bacteria and fungi. CRISPR/Cas systems can target plasmids or the bacterial chromosome of AMR-bacteria, and it is especially necessary to have an efficient entry into the target cells, which can be achieved through nanoparticles or bacteriophages. Regarding antibiotic development and production, though the use of CRISPR/Cas in this field is still modest, there is an untapped reservoir of bacterial and fungal natural products, with over 95% yet to be characterized. In Streptomyces, a key antibiotic-producing bacterial genus, CRISPR/Cas has been successfully used to activate silent biosynthetic gene clusters, leading to the discovery of new antibiotics. CRISPR/Cas is also applicable to non-model bacteria and different species of fungi, making it a versatile tool for natural products discovery. Moreover, CRISPR/Cas-based studies offer insights into metabolic regulation and biosynthetic pathways in both bacteria and fungi, highlighting its utility in understanding genetic regulation and improving industrial strains. In this work, we review ongoing innovations on ways to treat antimicrobial resistances and on antibiotic discovery using CRISPR/Cas platforms, highlighting the role of bacteria and fungi in these processes.}, }
@article {pmid39766302, year = {2024}, author = {Middlezong, W and Stinnett, V and Phan, M and Phan, B and Morsberger, L and Klausner, M and Ghabrial, J and DeMetrick, N and Zhu, J and James, T and Pallavajjala, A and Gocke, CD and Baer, MR and Zou, YS}, title = {Rapid Detection of PML::RARA Fusions in Acute Promyelocytic Leukemia: CRISPR/Cas9 Nanopore Sequencing with Adaptive Sampling.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766302}, issn = {2218-273X}, support = {ATIP grant//Johns Hopkins Institute for Clinical and Translational Research (ICTR)/ ; }, mesh = {Humans ; *Leukemia, Promyelocytic, Acute/genetics/diagnosis ; *Oncogene Proteins, Fusion/genetics ; *Nanopore Sequencing/methods ; *CRISPR-Cas Systems/genetics ; Retinoic Acid Receptor alpha/genetics ; Promyelocytic Leukemia Protein/genetics ; }, abstract = {Acute promyelocytic leukemia (APL) accounts for approximately 10-15% of newly diagnosed acute myeloid leukemia cases and presents with coagulopathy and bleeding. Prompt diagnosis and treatment are required to minimize early mortality in APL as initiation of all-trans retinoic acid therapy rapidly reverses coagulopathy. The PML::RARA fusion is a hallmark of APL and its rapid identification is essential for rapid initiation of specific treatment to prevent early deaths from coagulopathy and bleeding and optimize patient outcomes. Given limitations and long turnaround time of current gene fusion diagnostic strategies, we have developed a novel amplification-free nanopore sequencing-based approach with low cost, easy setup, and fast turnaround time. We termed the approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). Using CENAS, we successfully sequenced breakpoints of typical and atypical PML::RARA fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting PML::RARA fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical PML::RARA fusions and identified additional genes (ANKFN1 and JOSD1) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the ANKFN1 gene, the JOSD1 gene, and the 13q14.13 genomic region flanking with the SIAH3 and ZC3H13 genes have not been reported in the atypical PML::RARA fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a PML::RARA fusion. Given the early death rate in APL patients still reaches 15%, and ~10% of APL patients are resistant to initial therapy or prone to relapse, further sequencing studies of typical and atypical PML::RARA fusion might shed light on the pathophysiology of the disease and its responsiveness to treatment. Understanding the involvement of additional genes and positional effects related to the PML and RARA genes could shed light on their role in APL and may aid in the development of novel targeted therapies.}, }
@article {pmid39766265, year = {2024}, author = {Hirose, J and Aizawa, E and Yamamoto, S and Xu, M and Iwai, S and Suzuki, K}, title = {Lipid Nanoparticles Enable Efficient In Vivo DNA Knock-In via HITI-Mediated Genome Editing.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766265}, issn = {2218-273X}, support = {21H04811//The Japan Society for the Promotion of Science KAKENHI/ ; 22K19405//The Japan Society for the Promotion of Science KAKENHI/ ; JP23ek0109521//Japan Agency for Medical Research and Development/ ; N.A.//Daiichi Sankyo Foundation of Life Science/ ; N.A.//Osaka University Honors Program for Graduate Schools in Science, Engineering and Informatics/ ; JPMXP1224 OS1059//Ministry of Education, Culture, Sports, Science and Technology/ ; }, mesh = {*Gene Editing/methods ; Animals ; *Nanoparticles/chemistry ; Mice ; *Gene Knock-In Techniques ; *Lipids/chemistry ; *DNA/genetics/administration & dosage ; Humans ; Liver/metabolism ; CRISPR-Cas Systems ; Green Fluorescent Proteins/genetics/metabolism ; Liposomes ; }, abstract = {In vivo genome editing holds great therapeutic potential for treating monogenic diseases by enabling precise gene correction or addition. However, improving the efficiency of delivery systems remains a key challenge. In this study, we investigated the use of lipid nanoparticles (LNPs) for in vivo knock-in of ectopic DNA. Our in vitro experiments demonstrated that the homology-independent targeted integration (HITI)-mediated genome-editing method achieved significantly higher knock-in efficiency at the Alb locus in hepatic cells compared to the traditional homology-directed repair (HDR)-mediated approach. By optimizing LNP composition and administration routes, we successfully achieved HITI-mediated GFP knock-in (2.1-2.7%) in the livers of mice through intravenous delivery of LNP-loaded genome editing components. Notably, repeated intravenous dosing led to a twofold increase in liver GFP knock-in efficiency (4.3-7.0%) compared to a single dose, highlighting the potential for cumulative genome editing effects. These findings provide a solid foundation for the use of LNPs in in vivo knock-in strategies, paving the way for future genome-editing therapies.}, }
@article {pmid39766243, year = {2024}, author = {Lopez-Barbera, A and Abasolo, N and Torrell, H and Canela, N and Fernández-Arroyo, S}, title = {Integrative Transcriptomic and Target Metabolite Analysis as a New Tool for Designing Metabolic Engineering in Yeast.}, journal = {Biomolecules}, volume = {14}, number = {12}, pages = {}, pmid = {39766243}, issn = {2218-273X}, support = {ACCIÓ-Eurecat TRAÇA 2023-FEREMIC//Agencia per a la Competitivit de l'Empresa/ ; SGR01275-IOMICS//Agència de Gestió d'Ajuts Universitaris i de Recerca/ ; Vicente López to A.L-B.//Fundació Eurecat/ ; }, mesh = {*Metabolic Engineering/methods ; *Saccharomyces cerevisiae/metabolism/genetics ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression Profiling/methods ; Terpenes/metabolism ; Metabolomics/methods ; }, abstract = {Precision fermentation processes, especially when using edited microorganisms, demand accuracy in the bioengineering process to maximize the desired outcome and to avoid adverse effects. The selection of target sites to edit using CRISPR/Cas9 can be complex, resulting in non-controlled consequences. Therefore, the use of multi-omics strategies can help in the design, selection and efficiency of genetic editing. In this study, we present a multi-omics approach based on targeted metabolite analysis and transcriptomics for the designing of CRISPR/Cas9 in baker's yeast as a more efficient strategy to select editing regions. Multi-omics shows potential to reveal new metabolic bottlenecks and to elucidate new metabolic fluxes, which could be a key factor in minimizing the metabolic burden in edited microorganisms. In our model, we focus our attention on the isoprenoid synthesis due to their industrial interest. Targeted metabolite detection combined with a transcriptomic analysis revealed hydroxymethylglutaryl-CoA reductases (HMGs) as the best target gene to induce an increase in isoprenoid synthesis. Thus, an extra copy of HMG1 was introduced using, for the first time, the synthetic UADH1 promoter. The multi-omics analysis of the recombinant strain results in an accurate assessment of yeast behavior during the most important growth phases, highlighting the metabolic burden, Crabtree effect or the diauxic shift during culture.}, }
@article {pmid39764496, year = {2025}, author = {Tamaki, M and Chiku, T and Suzuki, S and Misaki, A and Funakubo, A and Matsushima, Y and Yokota, K and Igimi, S and Kajikawa, A}, title = {Application of the SpCas9 inhibitor BRD0539 for CRISPR/Cas9-based genetic tools in Lacticaseibacillus paracasei.}, journal = {Bioscience of microbiota, food and health}, volume = {44}, number = {1}, pages = {70-79}, pmid = {39764496}, issn = {2186-6953}, abstract = {Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of Streptococcus pyogenes Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of Lacticaseibacillus paracasei, as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the sirA gene of L. paracasei. Then BRD0539 was used for CRISPR-dependent DNA cleavage in vivo. Our results suggested that the inhibitor worked partially in both Escherichia coli and L. paracasei. Next, we designed a CRISPRi system in a L. paracasei strain by inserting an inactive SpCas9 gene into the chromosome and introducing a plasmid encoding for a single guide RNA (sgRNA) targeting the sirA gene. Expression of sirA was successfully inhibited in the recombinant strains, and CRISPRi was abolished in an inhibitor-dependent manner. Our findings may help expand the CRISPR toolbox for research on lactic acid bacteria and other microbes.}, }
@article {pmid39762743, year = {2025}, author = {Shelenkov, A and Slavokhotova, A and Mikhaylova, Y and Akimkin, V}, title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Klebsiella pneumoniae.}, journal = {BMC microbiology}, volume = {25}, number = {1}, pages = {3}, pmid = {39762743}, issn = {1471-2180}, support = {075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Klebsiella pneumoniae/genetics/drug effects/pathogenicity ; *Plasmids/genetics ; *Virulence Factors/genetics ; *Genome, Bacterial/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Klebsiella Infections/microbiology/epidemiology ; Replicon/genetics ; Multilocus Sequence Typing ; Anti-Bacterial Agents/pharmacology ; Databases, Genetic ; Genomics ; }, abstract = {BACKGROUND: The infections of bacterial origin represent a significant problem to the public healthcare worldwide both in clinical and community settings. Recent decade was marked by limiting treatment options for bacterial infections due to growing antimicrobial resistance (AMR) acquired and transferred by various bacterial species, especially the ones causing healthcare-associated infections, which has become a dangerous issue noticed by the World Health Organization. Numerous reports shown that the spread of AMR is often driven by several species-specific lineages usually called the 'global clones of high risk'. Thus, it is essential to track the isolates belonging to such clones and investigate the mechanisms of their pathogenicity and AMR acquisition. Currently, the whole genome-based analysis is more and more often used for these purposes, including the epidemiological surveillance and analysis of mobile elements involved in resistance transfer. However, in spite of the exponential growth of available bacterial genomes, their representation usually lack uniformity and availability of supporting metadata, which creates a bottleneck for such investigations.
DESCRIPTION: In this database, we provide the results of a thorough genomic analysis of 61,857 genomes of a highly dangerous bacterial pathogen Klebsiella pneumoniae. Important isolate typing information including multilocus sequence typing (MLST) types (STs), assignment of the isolates to known global clones, capsular (KL) and lipooligosaccharide (O) types, the presence of CRISPR-Cas systems, and cgMLST profiles are given, and the information regarding the presence of AMR, virulence genes and plasmid replicons within the genomes is provided.
CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.11069018 . The database will facilitate selection of the proper reference isolate sets for any types of genome-based investigations. It will be helpful for investigations in the field of K. pneumoniae genomic epidemiology, as well as antimicrobial resistance analysis and the development of prevention measures against this important pathogen.}, }
@article {pmid39762363, year = {2025}, author = {Kang, GH and Ko, Y and Lee, JM}, title = {Enhancing virus-mediated genome editing for cultivated tomato through low temperature.}, journal = {Plant cell reports}, volume = {44}, number = {1}, pages = {22}, pmid = {39762363}, issn = {1432-203X}, support = {RS-2024-00322217//New Breeding Technologies Development Program, Rural Development Administration/ ; 2021R1A2C2093789//National Research Foundation of Korea/ ; }, mesh = {*Solanum lycopersicum/genetics/virology ; *Gene Editing/methods ; *Cold Temperature ; *Plant Viruses/genetics ; Potexvirus/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Secoviridae/genetics ; Plant Breeding/methods ; Oxidoreductases/genetics ; Plant Leaves/genetics/virology ; }, abstract = {Viral vector-mediated gene editing is enhanced for cultivated tomato under low temperature conditions, enabling higher mutation rates, heritable, and virus-free gene editing for efficient breeding. The CRISPR/Cas system, a versatile gene-editing tool, has revolutionized plant breeding by enabling precise genetic modifications. The development of robust and efficient genome-editing tools for crops is crucial for their application in plant breeding. In this study, we highly improved virus-induced genome-editing (VIGE) system for cultivated tomato. Vectors of tobacco rattle virus (TRV) and potato virus X (PVX) were used to deliver sgRNA targeting phytoene desaturase (SlPDS), along with mobile RNA sequences of tFT or tRNA[Ileu], into Cas9-overexpressing cultivated tomato (S. lycopersicum cv. Moneymaker). Our results demonstrate that low temperature significantly enhanced viral vector-mediated gene editing efficiency in both cotyledons and systemic upper leaves. However, no mutant progeny was obtained from TRV- and PVX301-infected MM-Cas9 plants. To address this challenge, we employed tissue culture techniques and found that low-temperature incubations at the initiation stage of tissue culture lead to enhanced editing efficiency in both vectors, resulting in a higher mutation rate (> 70%) of SlPDS in regenerated plants. Heritable gene-edited and virus-free progenies were successfully identified. This study presents a straightforward approach to enhance VIGE efficiency and the expeditious production of gene-edited lines in tomato breeding.}, }
@article {pmid39762235, year = {2025}, author = {Hollingsworth, EW and Liu, TA and Alcantara, JA and Chen, CX and Jacinto, SH and Kvon, EZ}, title = {Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {409}, pmid = {39762235}, issn = {2041-1723}, support = {T32 GM008620/GM/NIGMS NIH HHS/United States ; S10 OD021718/OD/NIH HHS/United States ; T32 NS082174/NS/NINDS NIH HHS/United States ; F30 HD110233/HD/NICHD NIH HHS/United States ; S10 OD010794/OD/NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; S10 RR025496/RR/NCRR NIH HHS/United States ; R01 HD115268/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; *Otx Transcription Factors/genetics/metabolism ; MicroRNAs/genetics/metabolism ; Single-Cell Analysis ; Autism Spectrum Disorder/genetics/metabolism ; Craniofacial Abnormalities/genetics ; Genes, Reporter ; Female ; CRISPR-Cas Systems ; Genetic Variation ; Brain/metabolism ; Male ; Alleles ; }, abstract = {Functional analysis of non-coding variants associated with congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.}, }
@article {pmid39761113, year = {2025}, author = {Kardailsky, A and Durán-Vinet, B and Nester, G and Ayad, ME and Raes, EJ and Jeunen, GJ and Miller, AK and McVey, P and Corrigan, S and Fraser, M and Goncalves, P and Burnell, S and Bennett, A and Rauschert, S and Bayer, PE}, title = {Monitoring the Land and Sea: Enhancing Efficiency Through CRISPR-Cas Driven Depletion and Enrichment of Environmental DNA.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2024.0050}, pmid = {39761113}, issn = {2573-1602}, abstract = {Characterizing biodiversity using environmental DNA (eDNA) represents a paradigm shift in our capacity for biomonitoring complex environments, both aquatic and terrestrial. However, eDNA biomonitoring is limited by biases toward certain species and the low taxonomic resolution of current metabarcoding approaches. Shotgun metagenomics of eDNA enables the collection of whole ecosystem data by sequencing all molecules present, allowing characterization and identification. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins (Cas)-based methods have the potential to improve the efficiency of eDNA metagenomic sequencing of low-abundant target organisms and simplify data analysis by enrichment of target species or nontarget DNA depletion before sequencing. Implementation of CRISPR-Cas in eDNA has been limited due to a lack of interest and support in the past. This perspective synthesizes current approaches of CRISPR-Cas to study underrepresented taxa and advocate for further application and optimization of depletion and enrichment methods of eDNA using CRISPR-Cas, holding promise for eDNA biomonitoring.}, }
@article {pmid39760503, year = {2025}, author = {Zhu, Z and Lu, S and Wang, H and Wang, F and Xu, W and Zhu, Y and Xue, J and Yang, L}, title = {Innovations in Transgene Integration Analysis: A Comprehensive Review of Enrichment and Sequencing Strategies in Biotechnology.}, journal = {ACS applied materials & interfaces}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsami.4c14208}, pmid = {39760503}, issn = {1944-8252}, abstract = {Understanding the integration of transgene DNA (T-DNA) in transgenic crops, animals, and clinical applications is paramount for ensuring the stability and expression of inserted genes, which directly influence desired traits and therapeutic outcomes. Analyzing T-DNA integration patterns is essential for identifying potential unintended effects and evaluating the safety and environmental implications of genetically modified organisms (GMOs). This knowledge is crucial for regulatory compliance and fostering public trust in biotechnology by demonstrating transparency in genetic modifications. This review highlights recent advancements in T-DNA integration analysis, specifically focusing on targeted DNA enrichment and sequencing strategies. We examine key technologies, such as polymerase chain reaction (PCR)-based methods, hybridization capture, RNA/DNA-guided endonuclease-mediated enrichment, and high-throughput resequencing, emphasizing their contributions to enhancing precision and efficiency in transgene integration analysis. We discuss the principles, applications, and recent developments in these techniques, underscoring their critical role in advancing biotechnological products. Additionally, we address the existing challenges and future directions in the field, offering a comprehensive overview of how innovative DNA-targeted enrichment and sequencing strategies are reshaping biotechnology and genomics.}, }
@article {pmid39760089, year = {2024}, author = {Li, K and Luo, T and Zhang, Y and Li, C and Chen, H and Xia, C and Gao, C}, title = {Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1469558}, pmid = {39760089}, issn = {2235-2988}, mesh = {*Mycoplasma hyopneumoniae/genetics/isolation & purification ; *CRISPR-Cas Systems ; Animals ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia of Swine, Mycoplasmal/diagnosis/microbiology ; *Recombinases/metabolism/genetics ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Molecular Diagnostic Techniques/methods ; Limit of Detection ; }, abstract = {Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in porcine respiratory disease complex, and circulates in the swine industry worldwide. The prevention and control of M. hyopneumoniae is complicated. Thus, a recombinase-aided amplification (RAA) assay coupled with the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas12a system was established for the detection of M. hyopneumoniae. The most suitable primer pairs and CRISPR RNA (crRNA) were screened and selected for the RAA-CRISPR/Cas12a detection system. We have achieved a detection limit of 1 copy/µL and 5 copies/µL per reaction for the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a-lateral flow assay (LFA), respectively. Furthermore, the RAA-CRISPR/Cas12a system displayed no cross-reactivity with other respiratory pathogens. The performance of the RAA-CRISPR/Cas12a system was compared with PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry-exit inspection and quarantine industry standard (SN/T4104-2015) for clinical samples, and good consistency with these methods was observed. Above all, the methods shed a light on the convenient, portable, visual, highly sensitive and specific detection of M. hyopneumoniae, demonstrating a great application potential for on-site monitoring of M. hyopneumoniae in the field.}, }
@article {pmid39759869, year = {2024}, author = {Gong, Z and Wang, W and Zhao, Y and Wang, Y and Sun, R and Zhang, H and Wang, F and Lu, Y and Zhang, J}, title = {Analysis of the pathogenicity and pathological characteristics of NOTCH3 gene-sparing cysteine mutations in vitro and in vivo models.}, journal = {Frontiers in molecular neuroscience}, volume = {17}, number = {}, pages = {1391040}, pmid = {39759869}, issn = {1662-5099}, abstract = {BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common inherited cerebral small vessel diseases caused by the NOTCH3 gene mutation. This mutation leads to the accumulation of NOTCH3 extracellular domain protein (NOTCH3[ECD]) into the cerebral arterioles, causing recurrent stroke, white matter lesions, and cognitive impairment. With the development of gene sequencing technology, cysteine-sparing mutations can also cause CADASIL disease, however, the pathogenicity and pathogenic mechanisms of cysteine-sparing mutations remain controversial.
OBJECTIVE: To analyze the pathogenicity and pathological features of cysteine-sparing mutations in both in vitro and in vivo mouse models.
METHODS: A cysteine-sparing mutant of NOTCH3[ECD] R75Q was constructed by lentiviral transfection in vitro, and the NOTCH3 R75Q knock-in mouse model was constructed by CRISPR/Cas-mediated genome engineering in vivo. A cycloheximide pulse-chase experiment was used to analyze the degradation of NOTCH3 extracellular domain proteins, and the deposition characteristics of NOTCH3[ECD] were quantitatively analyzed by immunohistochemical staining. The characteristics of the smooth muscle cells and granular osmiophilic materials were observed using electron microscopy.
RESULTS: We elucidated that the NOTCH3 R75Q mutation is pathogenic. NOTCH3[ECD] R75Q was found to be resistant to protein degradation and more likely to cause abnormal aggregation of NOTCH3[ECD], resulting in reduced cell activity in vitro. The NOTCH3 R75Q mouse model showed pathological characteristics of CADASIL, with age-dependent NOTCH3[ECD], granular osmiophilic material, and degenerated smooth muscle cells detected in the brain.
CONCLUSION: To our knowledge, this is the first study to analyze the pathogenicity of NOTCH3 R75Q cysteine-sparing mutations in both in vitro and in vivo models. We demonstrate that NOTCH3[ECD] induced by NOTCH3 R75Q mutation has toxic effects on cells and reveal the deposition characteristics of NOTCH3[ECD] in the brain. This provides a feasible model and lays the foundation for further studies on the pathogenesis and therapeutic strategies of NOTCH3 cysteine-sparing mutations.}, }
@article {pmid39758988, year = {2024}, author = {Wu, X and Xiang, R and Yang, D and He, X and Zhu, L and Sun, F and Li, H and Pi, N and Li, Y}, title = {HRP-integrated CRISPR-Cas12a biosensor for rapid point-of-care detection of Langya henipavirus.}, journal = {iScience}, volume = {27}, number = {12}, pages = {111466}, pmid = {39758988}, issn = {2589-0042}, abstract = {Global pandemic has emphasized the needs for advanced pathogen diagnosis in dealing with newly emerged infectious threats, including the Langya henipavirus (LayV). LayV, as an emerging zoonotic pathogen, has potential to cause pandemic, but lacks of rapid diagnostic tools, particularly at point-of-care level. Here, we leveraged the merits of CRISPR-Cas12a biosensing and established a highly sensitive LayV detection method. By integrating CRISPR-Cas12a with RPA, 10 copies/μL ultra-sensitive LayV RNA detection has been achieved at room temperature within 30 min. More importantly, this study developed a special horseradish peroxidase (HRP)-single-stranded DNA (ssDNA) reporter enabling CRISPR-Cas12a to detect LayV RNA without pre-amplification, achieving a sensitivity of 1,200 copies/μL detectable by the naked eye. These explorations can serve as accelerator for CRISPR-Cas biosensing toward rapid response for newly emerged biological threats, and also provide a method to realize simple, precise, and amplicon-free point-of-care pathogen screening for resource limited or underdevelopment regions.}, }
@article {pmid39756423, year = {2025}, author = {Haeusser, LA and Becker, H and Kuhlburger, L and Zago, M and Walter, B and Tsiami, F and Erdmann, S and Trampert, J and Surender, S and Stahl, A and Templin, M and Wegner, E and Schmidt, T and Schmees, C and Casadei, N and Sevenich, L and Claassen, M and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G}, title = {Genome-wide CRISPR-Cas 9 screens identify BCL family members as modulators of response to regorafenib in experimental glioma.}, journal = {Neuro-oncology}, volume = {}, number = {}, pages = {}, doi = {10.1093/neuonc/noae278}, pmid = {39756423}, issn = {1523-5866}, abstract = {BACKGROUND: Registered systemic treatment options for glioblastoma patients are limited. The phase II REGOMA trial suggested an improvement of median overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE. So far, regorafenib has been administered as monotherapy or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib might be a reasonable strategy. Here, we aimed at identifying functionally-instructed combination therapies involving regorafenib.
METHODS: We applied a genome-wide CRISPR-Cas9-based functional genomics target discovery approach using activation and knockout screens followed by genetic, pharmacological, functional validations. Regorafenib-induced molecular alterations were assessed by RNAsequencing and DigiWest. We investigated selected functionally-instructed combination therapies in three orthotopic glioma mouse models in vivo (syngeneic SMA560/VM/Dk model and two xenograft models) and performed immunohistochemistry of post-treatment brains.
RESULTS: We identified potential modifiers of regorafenib response including BCL2, BCL2L1, ITGB3, FOXC1, SERAC1, ARAF, and PLCE1. The combination of regorafenib with Bcl-2/Bcl-xL inhibition was superior to both monotherapies alone in vitro, ex vivo and in vivo. We identified regorafenib-induced regulations of the Bcl-2 downstream target chemokine receptor 1 (CCR1) as one potential underlying molecular mediator. Furthermore, regorafenib led to changes in the myeloid compartment of the glioma-associated microenvironment.
CONCLUSION: This preclinical study uses a functional genomics-based target discovery approach with subsequent validations involving regorafenib. It serves as a biological rationale for clinical translation. Particularly, an investigation of the combination of regorafenib plus navitoclax within a clinical trial is warranted.}, }
@article {pmid39755699, year = {2025}, author = {Halegua, T and Risson, V and Carras, J and Rouyer, M and Coudert, L and Jacquier, A and Schaeffer, L and Ohlmann, T and Mangeot, PE}, title = {Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {397}, pmid = {39755699}, issn = {2041-1723}, mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; Ribonucleoproteins/metabolism/genetics ; Hematopoietic Stem Cells/metabolism/cytology/virology ; Myoblasts/metabolism/cytology ; Virion/metabolism/genetics ; Stem Cells/metabolism/cytology ; }, abstract = {Prime Editing can rewrite genes in living cells by allowing point mutations, deletions, or insertion of small DNA sequences with high precision. However, its safe and efficient delivery into human stem cells remains a technical challenge. In this report, we engineer Nanoscribes, virus-like particles that encapsidate ribonucleoprotein complexes of the Prime Editing system and allow their delivery into recipient cells. We identify key features that unlock the potential of Nanoscribes, including the use of multiple fusogens, the improvement of pegRNAs structures, their encoding by a Pol II system and the optimization of Prime-Editors. Nanoscribes edit HEK293T with an efficiency of 68% at the HEK3 locus with increased fidelity over DNA-transfection and support pegRNA-multiplexing. Importantly, Nanoscribes permit editing of myoblasts, hiPSCs and hiPSCs-derived hematopoietic stem cells with an editing efficiency up to 25%. Nanoscribes is an asset for development of next generation genome editing approaches using VLPs.}, }
@article {pmid39754035, year = {2025}, author = {Xiang, T and Feng, H and Xing, XH and Zhang, C}, title = {GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.}, journal = {BMC bioinformatics}, volume = {26}, number = {1}, pages = {1}, pmid = {39754035}, issn = {1471-2105}, support = {21938004//National Natural Science Foundation of China/ ; U2032210//National Natural Science Foundation of China/ ; 2023YFC3402300//National Key Research and Development Program of China/ ; }, mesh = {*Internet ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Software ; Prokaryotic Cells/metabolism ; Genomics/methods ; Genome, Bacterial ; }, abstract = {BACKGROUND: CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications.
RESULTS: We introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. It also provides the capability to design sgRNAs for newly discovered organisms by accepting uploaded design resource. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system.
CONCLUSIONS: We present a web server that allows the construction of genome-scale CRISPRi sgRNA libraries for prokaryotes. It mitigates off-target effects through a robust quality control framework, leveraging prior experimental knowledge within an end-to-end, user-friendly pipeline.}, }
@article {pmid39753078, year = {2024}, author = {Zhang, Y and Dong, X and Jiang, C and Yu, Y and Zhang, H and Fu, J and Su, G and Liu, Y}, title = {A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127491}, doi = {10.1016/j.talanta.2024.127491}, pmid = {39753078}, issn = {1873-3573}, abstract = {Tetracycline (TC) is widely used in veterinary medicine and animal feed; however, TC residues in food pose a risk to human health. Thus, the sensitive and selective detection of TC is needed to ensure food safety. Herein, we developed a CRISPR-Cas12a biosensor with competitive aptamer binding to detect TC residues. The aptasensor, formed by hybridizing activator DNA with TC-specific aptamers on streptavidin-modified magnetic beads, releases activator DNA in a TC concentration-dependent manner. This activated the Cas12a-crRNA complex, which cleaved single-strand DNA reporters to generate a detectable fluorescence signal. The TC signal was amplified through a two-step incubation reaction, with a detection limit as low as 9.45 × 10[-5] μg L[-1]. The assay showed high selectivity and good recovery rates in various biological samples (e.g., honey, milk, fish), demonstrating the applicability of the biosensors in pollutant detection.}, }
@article {pmid39752253, year = {2025}, author = {Mutte, SK and Barendse, P and Ugarte, PB and Swarts, DC}, title = {Distribution of bacterial DNA repair proteins and their co-occurrence with immune systems.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115110}, doi = {10.1016/j.celrep.2024.115110}, pmid = {39752253}, issn = {2211-1247}, abstract = {Bacteria encode various DNA repair pathways to maintain genome integrity. However, the high degree of homology between DNA repair proteins or their domains hampers accurate identification. Here, we describe a stringent search strategy to identify DNA repair proteins and provide a systematic analysis of taxonomic distribution and co-occurrence of DNA repair proteins involved in RecA-dependent homologous recombination. Our results reveal the widespread presence of RecA, SSB, and RecOR proteins and phyla-specific distribution for the DNA repair complexes RecBCD, AddAB, and AdnAB. Furthermore, we report co-occurrences of DNA repair proteins with immune systems, including specific CRISPR-Cas subtypes, prokaryotic Argonautes (pAgos), dGTPases, GAPS2, and Wadjet. Our results imply that while certain DNA repair proteins and immune systems might function in conjunction, no immune system strictly relies on a specific DNA repair protein. As such, these findings offer an updated perspective on the distribution of DNA repair systems and their connection to immune systems in bacteria.}, }
@article {pmid39752243, year = {2025}, author = {Riaz, A and Uzair, M and Raza, A and Inam, S and Iqbal, R and Jameel, S and Bibi, B and Khan, MR}, title = {Enhancing the productivity and resilience of rice (Oryza sativa) under environmental stress conditions using clustered regularly interspaced short palindromic repeats (CRISPR) technology.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP24101}, pmid = {39752243}, issn = {1445-4416}, mesh = {*Oryza/genetics ; *Stress, Physiological/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plants, Genetically Modified/genetics ; }, abstract = {Rice (Oryza sativa) is a crucial staple crop worldwide, providing nutrition to more than half of the global population. Nonetheless, the sustainability of grain production is increasingly jeopardized by both biotic and abiotic stressors exacerbated by climate change, which increases the crop's rvulnerability to pests and diseases. Genome-editing by clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR-Cas9) presents a potential solution for enhancing rice productivity and resilience under climatic stress. This technology can alter a plant's genetic components without the introduction of foreign DNA or genes. It has become one of the most extensively used approaches for discovering new gene functions and creating novel varieties that exhibit a higher tolerance to both abiotic and biotic stresses, herbicide resistance, and improved yield production. This study examines numerous CRISPR-Cas9-based genome-editing techniques for gene knockout, gene knock-in, multiplexing for simultaneous disruption of multiple genes, base-editing, and prime-editing. This review elucidates the application of genome-editing technologies to enhance rice production by directly targeting yield-related genes or indirectly modulating numerous abiotic and biotic stress-responsive genes. We highlight the need to integrate genetic advancements with conventional and advanced agricultural methods to create rice varieties that are resilient to stresses, thereby safeguarding food security and promoting agricultural sustainability amid climatic concerns.}, }
@article {pmid39749289, year = {2024}, author = {Bhoobalan-Chitty, Y and Stouf, M and De Paepe, M}, title = {Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1495968}, pmid = {39749289}, issn = {2673-3439}, abstract = {CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, α-gt and β-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.}, }
@article {pmid39748374, year = {2025}, author = {Wan, X and Kong, J and Hu, X and Liu, L and Yang, Y and Li, H and Liu, G and Niu, X and Chen, F and Zhang, D and Zhu, D and Zhang, Y}, title = {SiCLAT: simultaneous imaging of chromatin loops and active transcription in living cells.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {1}, pmid = {39748374}, issn = {1474-760X}, mesh = {Animals ; *Chromatin/metabolism ; Mice ; *Transcription, Genetic ; Enhancer Elements, Genetic ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Cell Differentiation ; }, abstract = {We present SiCLAT, which introduces a dCas9-dCas13d cassette into the mouse genome. This model enables the stable expression of both dCas9 and dCas13 proteins in diverse cell populations, facilitating concurrent labeling of DNA and RNA across various cell types. Using SiCLAT, we accurately labeled chromatin loop anchor interactions and associated gene transcription during myogenic differentiation. This imaging system offers a novel means of directly observing cis-element interactions and the corresponding gene transcription in living primary cells, thus providing real-time imaging for comprehensive mechanistic investigations of dynamic enhancer-promoter or enhancer-enhancer interactions in regulating transcription activation within living cells.}, }
@article {pmid39747875, year = {2025}, author = {Raghavan, R and Friedrich, MJ and King, I and Chau-Duy-Tam Vo, S and Strebinger, D and Lash, B and Kilian, M and Platten, M and Macrae, RK and Song, Y and Nivon, L and Zhang, F}, title = {Rational engineering of minimally immunogenic nucleases for gene therapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {105}, pmid = {39747875}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; CRISPR-Associated Protein 9/metabolism/genetics ; Protein Engineering/methods ; Proprotein Convertase 9/immunology/genetics/metabolism ; Mice ; HEK293 Cells ; T-Lymphocytes, Cytotoxic/immunology ; Epitopes/immunology ; Endonucleases/metabolism/genetics ; Female ; }, abstract = {Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.}, }
@article {pmid39747848, year = {2025}, author = {Escobar, H and Di Francescantonio, S and Smirnova, J and Graf, R and Müthel, S and Marg, A and Zhogov, A and Krishna, S and Metzler, E and Petkova, M and Daumke, O and Kühn, R and Spuler, S}, title = {Gene-editing in patient and humanized-mice primary muscle stem cells rescues dysferlin expression in dysferlin-deficient muscular dystrophy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {120}, pmid = {39747848}, issn = {2041-1723}, mesh = {*Dysferlin/genetics/metabolism ; Animals ; Humans ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; Frameshift Mutation ; CRISPR-Cas Systems ; Muscular Dystrophies/therapy/genetics/metabolism ; Exons/genetics ; Stem Cells/metabolism ; Female ; Membrane Proteins/genetics/metabolism ; Male ; Muscular Dystrophies, Limb-Girdle/therapy/genetics/metabolism ; Genetic Therapy/methods ; }, abstract = {Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulates the patients' phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.}, }
@article {pmid39747609, year = {2025}, author = {Motooka, Y and Tanaka, H and Maeda, Y and Katabuchi, M and Mashimo, T and Toyokuni, S}, title = {Heterozygous mutation in BRCA2 induces accelerated age-dependent decline in sperm quality with male subfertility in rats.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {447}, pmid = {39747609}, issn = {2045-2322}, support = {JP23K08883//Japan Society for the Promotion of Science/ ; JP19H0546//Japan Society for the Promotion of Science/ ; JP16H06276 [AdAMS (Aa210038)]//Japan Society for the Promotion of Science/ ; JPMJCR19H4//JST CREST/ ; }, mesh = {Male ; Animals ; *BRCA2 Protein/genetics ; Rats ; *Spermatozoa/metabolism ; *Infertility, Male/genetics ; Heterozygote ; Mutation ; Exons/genetics ; DNA Breaks, Double-Stranded ; Aging/genetics ; Humans ; Apoptosis/genetics ; Testis/pathology/metabolism ; CRISPR-Cas Systems ; }, abstract = {Tumor suppressor BRCA2 executes homologous recombination to repair DNA double-strand breaks in collaboration with RAD51, involving exon 11 and 27. Exon 11 constitutes a region where pathogenic variants (PVs) accumulate, and mutations in this region are known to contribute to carcinogenesis. However, the impact of the heterozygous PVs of BRCA2 exon 11 on the life quality beyond cancer risk, including male fertility, remains unclear. Here, we established a rat model with a frameshift on the seventh BRC repeat in Brca2 exon 11 (Brca2[+/p.T1942fs]), which is homologous to human BRCA2[+/p.T1974fs], using CRISPR/Cas9 system. Our analyses revealed that the heterozygous rats with the PV in the BRCA2 exon 11 showed increased DNA double-strand breaks and apoptosis in spermatogonia and spermatocytes, accelerated testicular germ cell loss, and deterioration in sperm quality according with aging, ultimately resulting in early male reproductive dysfunction. Of note, these alterations in testes and sperm, including DNA fragmentation in spermatozoa, were observed from completion of sexual maturation. The present findings suggest that it is crucial to consider not only cancer risk but also potential declines in reproductive capacity in men carrying BRCA2 exon 11 PVs. Further investigation is warranted to determine whether similar traits appear in humans.}, }
@article {pmid39747369, year = {2025}, author = {Patil, S and Siji, A and Mallur, D and Kruthika, BS and Gheewalla, N and Karve, S and Kavathekar, M and Tarai, B and Naik, M and Hegde, V and Rangineni, J and Gupta, V and Chandru, V and Pradeep, BE and Arora, R}, title = {PathCrisp: an innovative molecular diagnostic tool for early detection of NDM-resistant infections.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {490}, pmid = {39747369}, issn = {2045-2322}, support = {2021 HTH 018//Rockefeller Foundation Grant/ ; 2021 HTH 018//Rockefeller Foundation Grant/ ; }, mesh = {*beta-Lactamases/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Enterobacteriaceae Infections/diagnosis/microbiology ; Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification ; Sensitivity and Specificity ; Early Diagnosis ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; }, abstract = {The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time and guide therapy. PathCrisp assay, a combination of loop-mediated isothermal amplification and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the polymerase chain reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant enterobacteriaceae clinical samples. Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 h, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy.}, }
@article {pmid39747289, year = {2025}, author = {Frusteri Chiacchiera, A and Casanova, M and Bellato, M and Piazza, A and Migliavacca, R and Batt, G and Magni, P and Pasotti, L}, title = {Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {261}, pmid = {39747289}, issn = {2045-2322}, support = {1139857//Regione Lombardia/ ; 20-PAMR-0010 (Seq2DiAg)//Agence Nationale de la Recherche/ ; 59576 (2021)//Fondazione Cassa di Risparmio di Padova e Rovigo/ ; INCEPTION//Institut Pasteur/ ; }, mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; *Escherichia coli/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Mutation ; }, abstract = {The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.}, }
@article {pmid39747101, year = {2025}, author = {Lin, JF and Liu, ZX and Chen, DL and Huang, RZ and Cao, F and Yu, K and Li, T and Mo, HY and Sheng, H and Liang, ZB and Liao, K and Han, Y and Li, SS and Zeng, ZL and Gao, S and Ju, HQ and Xu, RH}, title = {Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {263}, pmid = {39747101}, issn = {2041-1723}, support = {82303306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82373376//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/metabolism/pathology ; *Drug Resistance, Neoplasm/genetics ; *NF-kappa B/metabolism ; *Cell Nucleus/metabolism ; Cell Line, Tumor ; *Glutamate-Cysteine Ligase/metabolism/genetics ; Phosphorylation ; Animals ; Antineoplastic Agents/therapeutic use/pharmacology ; Mice ; Gene Expression Regulation, Neoplastic ; p38 Mitogen-Activated Protein Kinases/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Active Transport, Cell Nucleus ; Mice, Nude ; }, abstract = {Metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. Here, utilizing a metabolic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout library screen, we observe that the loss of glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably increases the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B (NF-κB)-repressing factor (NKRF), to promote NF-κB activity and facilitate chemoresistance. In response to platinum drug treatment, GCLM is phosphorylated by P38 MAPK at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM and phospho-GCLM correlate with an unfavorable prognosis and poor benefit from standard chemotherapy. Overall, our work highlights the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance.}, }
@article {pmid39747086, year = {2025}, author = {Albuquerque-Wendt, A and McCoy, C and Neish, R and Dobramysl, U and Alagöz, Ç and Beneke, T and Cowley, SA and Crouch, K and Wheeler, RJ and Mottram, JC and Gluenz, E}, title = {TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {299}, pmid = {39747086}, issn = {2041-1723}, support = {221944/A/20/Z//Wellcome Trust (Wellcome)/ ; trans-LEISHion-EU FP7, No. 798736//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 211075/Z/18/Z//Wellcome Trust (Wellcome)/ ; 104111/Z/14/Z//Wellcome Trust (Wellcome)/ ; /WT_/Wellcome Trust/United Kingdom ; 15/16_MSD_836338//RCUK | Medical Research Council (MRC)/ ; 101064428 - LeishMOM//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (H2020 Excellent Science - Marie Skłodowska-Curie Actions)/ ; 200807/Z/16/Z//Wellcome Trust (Wellcome)/ ; UF160661//Royal Society/ ; ALTF 727-2021//European Molecular Biology Organization (EMBO)/ ; }, mesh = {Animals ; Mice ; *Gene Deletion ; *Macrophages/parasitology/metabolism ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; *Membrane Transport Proteins/genetics/metabolism ; Leishmania mexicana/genetics/metabolism ; Vacuolar Proton-Translocating ATPases/genetics/metabolism ; Female ; Mice, Inbred BALB C ; }, abstract = {For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H[+] ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes.}, }
@article {pmid39747083, year = {2025}, author = {Fei, J and Zhao, D and Pang, C and Li, J and Li, S and Qiao, W and Tan, J and Bi, C and Zhang, X}, title = {Mismatch prime editing gRNA increased efficiency and reduced indels.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {139}, pmid = {39747083}, issn = {2041-1723}, support = {32171449//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271483//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32225031//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022177//Youth Innovation Promotion Association of the Chinese Academy of Sciences (Youth Innovation Promotion Association CAS)/ ; }, mesh = {*Gene Editing/methods ; Humans ; *INDEL Mutation ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Base Pair Mismatch ; }, abstract = {Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA's 3' extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels.}, }
@article {pmid39747012, year = {2025}, author = {Tolosana, I and Willis, K and Gribble, M and Phillimore, L and Burt, A and Nolan, T and Crisanti, A and Bernardini, F}, title = {A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {206}, pmid = {39747012}, issn = {2041-1723}, support = {INV006610 "Target Malaria Phase II"//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; INV006610 "Target Malaria Phase II"//Bill and Melinda Gates Foundation (Bill & Melinda Gates Foundation)/ ; }, mesh = {*Anopheles/genetics ; Animals ; Female ; *Y Chromosome/genetics ; Male ; *Mosquito Vectors/genetics ; *Malaria/prevention & control/transmission ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; *Gene Editing/methods ; Genome, Insect ; }, abstract = {Genetic control - the deliberate introduction of genetic traits to control a pest or vector population - offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings, but hesitancy due to their invasive and persistent nature may delay their implementation. Here, instead, we describe a self-limiting strategy, designed to have geographically and temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. To our knowledge, our system represents a pioneering approach in the engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to seven times more efficient for population suppression than optimal versions of other self-limiting strategies, such as the widely used Sterile Insect Technique or the Release of Insects carrying a Dominant Lethal gene.}, }
@article {pmid39745650, year = {2025}, author = {Salvador-Martínez, I}, title = {Computational Methods for Lineage Reconstruction.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {355-373}, pmid = {39745650}, issn = {1940-6029}, mesh = {*Software ; *Cell Lineage/genetics ; *Algorithms ; *Phylogeny ; *Computational Biology/methods ; Animals ; CRISPR-Cas Systems ; Humans ; }, abstract = {The recent development of genetic lineage recorders, designed to register the genealogical history of cells using induced somatic mutations, has opened the possibility of reconstructing complete animal cell lineages. To reconstruct a cell lineage tree from a molecular recorder, it is crucial to use an appropriate reconstruction algorithm. Current approaches include algorithms specifically designed for cell lineage reconstruction and the repurposing of phylogenetic algorithms. These methods have, however, the same objective: to uncover the hierarchical relationships between cells and the sequence of cell divisions that have occurred during development. In this chapter, I will use the phylogenetic software FastTree to reconstruct a lineage tree, in a step-by-step manner, using data from a simulated CRISPR-Cas9 recorder. To ensure reproducibility, the code is presented as a Jupyter Notebook, available (together with the necessary input files) at https://github.com/irepansalvador/lineage_reconstruction_chapter .}, }
@article {pmid39745649, year = {2025}, author = {Espinosa-Medina, I}, title = {Design and Generation of TEMPO Reagents for Sequential Labeling and Manipulation of Vertebrate Cell Lineages.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {327-353}, pmid = {39745649}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish ; Mice ; *Cell Lineage ; CRISPR-Cas Systems ; Staining and Labeling/methods ; Luminescent Proteins/genetics/metabolism ; }, abstract = {During development, cells undergo a sequence of specification events to form functional tissues and organs. To investigate complex tissue development, it is crucial to visualize how cell lineages emerge and to be able to manipulate regulatory factors with temporal control. We recently developed TEMPO (Temporal Encoding and Manipulation in a Predefined Order), a genetic tool to label with different colors and genetically manipulate consecutive cell generations in vertebrates. TEMPO relies on CRISPR to activate a cascade of fluorescent proteins which can be imaged in vivo. Here, we explain the steps to design, generate, and express TEMPO constructs in zebrafish and mice.}, }
@article {pmid39745647, year = {2025}, author = {Raj, B}, title = {Single-Cell Profiling of Lineages and Cell Types in the Vertebrate Brain.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {299-310}, pmid = {39745647}, issn = {1940-6029}, mesh = {Animals ; *Single-Cell Analysis/methods ; *Brain/cytology/metabolism ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Cell Lineage/genetics ; *Gene Editing/methods ; Transcriptome ; Gene Expression Profiling/methods ; }, abstract = {CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.}, }
@article {pmid39745646, year = {2025}, author = {Bowling, S and Camargo, FD}, title = {CARLIN: A Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {281-298}, pmid = {39745646}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Cell Lineage/genetics ; *DNA Barcoding, Taxonomic/methods ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression/genetics ; }, abstract = {The CRISPR-activated repair lineage tracing (CARLIN) mouse line uses DNA barcoding to enable high-resolution tracing of cell lineages in vivo (Bowling et al, Cell 181, 1410-1422.e27, 2020). CARLIN mice contain expressed barcodes that allow simultaneous interrogation of lineage and gene expression information from single cells. Furthermore, barcode editing is fully inducible, resulting in cell lineage labeling that can be performed at any time point in development or adulthood. This chapter details the protocols followed for maintaining CARLIN mice, inducing barcoding, and amplifying the CARLIN barcode from DNA, RNA, and single-cell RNA-sequencing libraries for next-generation sequencing.}, }
@article {pmid39745643, year = {2025}, author = {Baron, CS and Alemany, A}, title = {Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {221-241}, pmid = {39745643}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish/genetics ; *Single-Cell Analysis/methods ; *DNA Barcoding, Taxonomic/methods ; *CRISPR-Cas Systems ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; Embryo, Nonmammalian/metabolism ; }, abstract = {ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.}, }
@article {pmid39745637, year = {2025}, author = {Gentile, E and Maynard, A and He, Z and Treutlein, B}, title = {Lineage Recording in Human Brain Organoids with iTracer.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {85-101}, pmid = {39745637}, issn = {1940-6029}, mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain/cytology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Lineage/genetics ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Transcriptome ; Cell Differentiation ; }, abstract = {Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enables highly resolved descriptions of cell states within these systems; however, approaches are needed to directly determine the lineage relationship between cells. Here we provide a detailed protocol (Fig. 1) for the application of iTracer (He Z, Maynard A, Jain A, et al., Nat Methods 19:90-99, 2022), a recently published lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. iTracer is used to explore clonality and lineage dynamics during brain organoid development. More broadly, iTracer can be adapted to any iPSC-derived culture system to dissect lineage dynamics during normal or perturbed development.}, }
@article {pmid39745602, year = {2025}, author = {Hajirnis, N}, title = {Re-arranging the Cis-regulatory Modules of Hox Complex in Drosophila via FLP-FRT and CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2889}, number = {}, pages = {11-24}, pmid = {39745602}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Drosophila melanogaster/genetics/growth & development ; Drosophila Proteins/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Genes, Homeobox/genetics ; Gene Editing/methods ; DNA Nucleotidyltransferases ; }, abstract = {FLP-FRT, a well-established technique for genome manipulation, and the revolutionary CRISPR/Cas9, known for its targeted indels, are combined in a novel approach. This unique method is applied to the Hox genes in the Drosophila melanogaster bithorax complex, which are closely located to the cis-regulatory modules that define their spatial-temporal regulation. The number and position of these genes are directly correlated to their expression pattern. This chapter unveils the exciting potential of this combinatorial use of FLP-FRT and CRISPR-Cas9 to rearrange the cis-regulatory modules of the Hox complex in Drosophila melanogaster.}, }
@article {pmid39745317, year = {2025}, author = {Hou, M and Yang, X and Gong, L and Shen, X}, title = {Surveillance of antimicrobial resistance using isothermal amplification: a review.}, journal = {Chemical communications (Cambridge, England)}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4cc05488a}, pmid = {39745317}, issn = {1364-548X}, abstract = {The monitoring of antibiotic resistance genes (ARGs) is crucial for understanding the level of antimicrobial resistance and the associated health burden, which in turn is essential for the control and prevention of antimicrobial resistance (AMR). Isothermal amplification, an emerging molecular biology technology, has been widely used for drug resistance detection. Furthermore, its compatibility with a range of technologies enables high-specificity, high-throughput, and portable and integrated detection in drug resistance, particularly in resource-limited areas. However, to date, reviews involved in isothermal amplification all concentrate on its technological advancements and its application in nucleic acid point-of-care testing. Few reviews have been published that focus specifically on the application of isothermal amplification in the detection of drug resistance. This review summarizes the detection principles of different isothermal amplification techniques and discusses their strengths and weaknesses as well as the applicable scenarios for drug resistance detection. It also summarizes advances in the application, challenges and prospects of isothermal amplification technologies in conjunction with different methods such as base mismatch, CRISPR-Cas, lateral flow immunoassay, sensing and microfluidic technologies for improvement of specificity, throughput and integration for drug resistance detection. It is anticipated that this review will assist scientists in comprehending the evolution of isothermal amplification in the context of drug resistance detection and provide insights into the prospective applications of isothermal amplification for highly integrated and immediate on-site detection of drug resistance.}, }
@article {pmid39744681, year = {2025}, author = {Park, M and Ryu, H and Heo, S and Kim, B and Park, J and Lim, KH and Han, SB and Park, H}, title = {Targeted demethylation of cathepsin D via epigenome editing rescues pathology in Alzheimer's disease mouse model.}, journal = {Theranostics}, volume = {15}, number = {2}, pages = {428-438}, pmid = {39744681}, issn = {1838-7640}, mesh = {Animals ; *Alzheimer Disease/genetics/therapy/metabolism ; *Disease Models, Animal ; Mice ; *Amyloid beta-Peptides/metabolism ; *Cathepsin D/metabolism/genetics ; *Gene Editing/methods ; *Mice, Transgenic ; *Epigenome ; Neurons/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; Humans ; Brain/metabolism/pathology ; Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Male ; }, abstract = {Background: Cathepsin D (Ctsd) has emerged as a promising therapeutic target for Alzheimer's disease (AD) due to its role in degrading intracellular amyloid beta (Aβ). Enhancing Ctsd activity could reduce Aβ42 accumulation and restore the Aβ42/40 ratio, offering a potential AD treatment strategy. Methods: This study explored Ctsd demethylation in AD mouse models using dCas9-Tet1-mediated epigenome editing. We identified dCas9-Tet1 as an effective tool for demethylating the endogenous Ctsd gene in primary neurons and in vivo brains. Results: Treatment with Ctsd-targeted dCas9-Tet1 in primary neurons overexpressing mutant APP (mutAPP) reduced Aβ peptide levels and the Aβ42/40 ratio. Additionally, in vivo demethylation of Ctsd via dCas9-Tet1 in 5xFAD mice significantly altered Aβ levels and alleviated cognitive and behavioral deficits. Conclusion: These findings offer valuable insights into developing epigenome editing-based gene therapy strategies for AD.}, }
@article {pmid39743757, year = {2024}, author = {Yu, CH and Lai, KL}, title = {Cure the Incurable: Update of Treatment in Inherited Neuromuscular Disorders.}, journal = {Acta neurologica Taiwanica}, volume = {33(4)}, number = {}, pages = {129-135}, pmid = {39743757}, issn = {1028-768X}, mesh = {Humans ; *Genetic Therapy/methods ; *Neuromuscular Diseases/therapy/genetics ; *CRISPR-Cas Systems ; }, abstract = {Originally thought to be incurable, huge therapeutic progress has been made in recent years in the field of inherited neuromuscular disorders. Approaches aiming to rescue the underlying pathophysiology, i.e. loss-of-function or gain-of-function mutations, have been developed via end-product replacement or gene delivery/modulation, with promising results. In the review, advanced treatment in some of the inherited neuromuscular disorders will be discussed. On the other hand, it has been found more than 1000 genes are responsible for the clinical diversities in this group of diseases, and finding a way which owns the therapeutic potential to various diseases is the optimal goal. The discovery of CRISPR-Cas9 system in the last decade offers such an opportunity and is under rigorous investigation. This important issue will also be discussed. Keywords: CRISPR-Cas9, gene therapy, gene delivery/modulation, inherited neuromuscular disorders, therapeutic advances.}, }
@article {pmid39743295, year = {2024}, author = {Jiang, Y and Pan, Q and Wang, Z and Lu, K and Xia, B and Chen, T}, title = {Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system.}, journal = {Journal of Zhejiang University. Science. B}, volume = {25}, number = {12}, pages = {1083-1096}, pmid = {39743295}, issn = {1862-1783}, support = {32273127, 31771648 and 31672653//the National Natural Science Foundation of China/ ; ZQ2020003//the Scientific Research Foundation of Jimei University/ ; }, mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Codon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Monophenol Monooxygenase/genetics ; CRISPR-Associated Protein 9/genetics ; RNA, Transfer/genetics ; Staphylococcus aureus/genetics ; PAX6 Transcription Factor/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.}, }
@article {pmid39743283, year = {2024}, author = {He, YM and Wu, ZP and He, J and Zhang, W and Zhu, FM}, title = {[Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {32}, number = {6}, pages = {1896-1902}, doi = {10.19746/j.cnki.issn.1009-2137.2024.06.040}, pmid = {39743283}, issn = {1009-2137}, mesh = {Humans ; HEK293 Cells ; *Gene Editing ; *Transfection ; *Hematopoietic Stem Cells ; RNA, Guide, CRISPR-Cas Systems ; beta 2-Microglobulin/genetics ; Histocompatibility Antigens Class I/genetics ; Flow Cytometry ; }, abstract = {OBJECTIVE: To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells.
METHODS: The easyedit small guide RNA(sgRNA) was designed according to the sequences of β2 microglobulin gene and synthesized by GenScript company. RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios (1∶1~1∶4). Control group and four transfection groups were performed respectively. HEK-293 cells and CD34[+] hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system. The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours, the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing.
RESULTS: The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34[+] hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours. There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing, indicating that sgRNA had obvious editing effect. In the transfection of HEK-293 cells, the highest proportion of HLA-I negative expression cells was (87.69±0.83)% when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1∶4. The cutting efficiency of T7E1 was the highest up to (38±2.0)% when the molar ratio was 1∶3. In the transfection of CD34[+] hematopoietic stem cells, the proportion of HLA-I negative expression cells was (91.56±3.39)% when the molar ratio was 1∶2, and the cutting efficiency of T7E1 was (64±8.45)% when the molar ratio was 1∶1.
CONCLUSION: This study provides an efficient gene editing method for classical HLA-I molecules, which can effectively silence the expression of class HLA-I molecules on the cell surface, and is suitable for stem cell system with difficult transfection.}, }
@article {pmid39741289, year = {2024}, author = {Yazdi, ZF and Roshannezhad, S and Sharif, S and Abbaszadegan, MR}, title = {Recent progress in prompt molecular detection of liquid biopsy using Cas enzymes: innovative approaches for cancer diagnosis and analysis.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1173}, pmid = {39741289}, issn = {1479-5876}, mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics/pathology ; *Neoplastic Cells, Circulating/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Cell-Free Nucleic Acids/blood ; Circulating Tumor DNA/blood ; }, abstract = {Creating fast, non-invasive, precise, and specific diagnostic tests is crucial for enhancing cancer treatment outcomes. Among diagnostic methods, those relying on nucleic acid detection are highly sensitive and specific. Recent developments in diagnostic technologies, particularly those leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are revolutionizing cancer detection, providing accurate and timely results. In clinical oncology, liquid biopsy has become a noninvasive and early-detectable alternative to traditional biopsies over the last two decades. Analyzing the nucleic acid content of liquid biopsy samples, which include Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), Circulating Cell-Free RNA (cfRNA), and tumor extracellular vesicles, provides a noninvasive method for cancer detection and monitoring. In this review, we explore how the characteristics of various Cas (CRISPR-associated) enzymes have been utilized in diagnostic assays for cancer liquid biopsy and highlight their main applications of innovative approaches in monitoring, as well as early and rapid detection of cancers.}, }
@article {pmid39741007, year = {2025}, author = {Lima, BA and Pais, AC and Dupont, J and Dias, P and Custódio, N and Sousa, AB and Carmo-Fonseca, M and Carvalho, C}, title = {Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells.}, journal = {Life science alliance}, volume = {8}, number = {3}, pages = {}, pmid = {39741007}, issn = {2575-1077}, mesh = {Humans ; *BRCA2 Protein/genetics/metabolism ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Gene Editing/methods ; DNA Repair/genetics ; Nonsense Mediated mRNA Decay/genetics ; RNA, Messenger/genetics/metabolism ; Cell Line, Tumor ; Female ; BRCA1 Protein/genetics/metabolism ; Rad51 Recombinase/genetics/metabolism ; Breast Neoplasms/genetics ; }, abstract = {Variants in the hereditary cancer-associated BRCA1 and BRCA2 genes can alter RNA splicing, producing transcripts that encode internally truncated yet potentially functional proteins. However, few studies have quantitatively analyzed variant-specific splicing isoforms. Here, we investigated cells heterozygous and homozygous for the BRCA2:c.681+5G>C variant. Using droplet digital RT-PCR, we identified two variant-specific mRNA isoforms. The predominant transcript is out-of-frame, contains a premature termination codon, and is degraded via the nonsense-mediated mRNA decay pathway. In addition, we detected a novel minor isoform encoding an internally truncated protein lacking non-essential domains. Homozygous mutant cells expressed low levels of BRCA2 protein and were defective in DNA repair. Using CRISPR-Cas9 gene editing, we induced the production of in-frame transcripts in mutant cells, which resulted in increased protein expression, enhanced RAD51 focus formation, and reduced chromosomal breaks after exposure to genotoxic agents. Our findings highlight the therapeutic potential of splicing modulation to restore BRCA2 function in mutant cells, offering a promising strategy to prevent cancer development.}, }
@article {pmid39740568, year = {2024}, author = {Zhan, X and Jiang, Y and Lei, J and Chen, H and Liu, T and Lan, F and Ying, B and Wu, Y}, title = {DNA Tetrahedron-enhanced single-particle counting integrated with cascaded CRISPR Program for ultrasensitive dual RNAs logic sensing.}, journal = {Journal of colloid and interface science}, volume = {683}, number = {Pt 2}, pages = {521-531}, doi = {10.1016/j.jcis.2024.12.182}, pmid = {39740568}, issn = {1095-7103}, abstract = {CRISPR-Cas-based technology, emerging as a leading platform for molecular assays, has been extensively researched and applied in bioanalysis. However, achieving simultaneous and highly sensitive detection of multiple nucleic acid targets remains a significant challenge for most current CRISPR-Cas systems. Herein, a CRISPR Cas12a based calibratable single particle counting-mediated biosensor was constructed for dual RNAs logic and ultra-sensitive detection in one tube based on DNA Tetrahedron (DTN)-interface supported fluorescent particle probes coupled with a novel synergistic cascaded strategy between CRISPR Cas13a system and strand displacement amplification (SDA). As expected, our platform enables dual RNA molecules intelligent detection using only one crRNA of Cas13a, achieving a sensitivity enhancement of three orders of magnitude assisted with multiple signal amplification and accurate fluorescence particle counting with DTN mediated nano-biointerface enhancement, compared to traditional bulk Cas13a assays. Moreover, the effectiveness and universality of our strategy are experimentally investigated and demonstrated through the detection of mRNAs (cervical cancer swab clinical samples and cultured cancer cells) and bacterial 16s rRNAs. This work not only proposes a highly promising avenue for designing CRISPR-based multiplex detection systems that excel in ultra-sensitivity, specificity, and clinical molecular diagnostics, but also provide new insights into the potential applications of nanotechnology in molecular diagnostics, functional surface engineering, and interface-mediated bioreactions.}, }
@article {pmid39740040, year = {2025}, author = {Cai, W and Yang, M and Zhao, Q and Yi, G and Lin, P and Chen, A and De, G}, title = {hURAT1 Transgenic Mouse Model for Evaluating Targeted Urate-Lowering Agents.}, journal = {International journal of rheumatic diseases}, volume = {28}, number = {1}, pages = {e70034}, doi = {10.1111/1756-185X.70034}, pmid = {39740040}, issn = {1756-185X}, support = {ZXKT23002//the Fundamental Research Funds for the Central Public Welfare Research Institutes/ ; 32273019//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Hyperuricemia/drug therapy/genetics/blood ; *Disease Models, Animal ; *Organic Anion Transporters/genetics/metabolism ; *Uric Acid/blood ; Humans ; *Organic Cation Transport Proteins/genetics/metabolism ; *Mice, Transgenic ; Uricosuric Agents/pharmacology/therapeutic use ; Gout/drug therapy/genetics/metabolism ; Mice, Inbred C57BL ; Male ; Gout Suppressants/pharmacology/therapeutic use ; Phenotype ; Gene Knock-In Techniques ; Hypoxanthine/metabolism ; Mice ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Urate transporter 1 (URAT1) is a well-known therapeutic target for reducing urate levels in the treatment of hyperuricemia and gout. However, current pharmacological studies have failed to evaluate the efficacy of URAT1 inhibitors in non-primate animal models. We established a human URAT1 (hURAT1) transgenic knock-in (KI) mouse model to assess uricosuric agents' effectiveness and characterize URAT1-caused pathogenesis.
METHODS: We generated hURAT1 transgenic mice using CRISPR/Cas9 KI technique. mUrat1 knockout was achieved by replacing exon 1 coding sequence with a human SLC22A12 coding sequence (CDS)-pA cassette. Based on the above transgenic mice, a hyperuricemia model was further established by hypoxanthine administration.
RESULTS: The hURAT1-KI mice successfully expressed hURAT1 protein to the apical side of the kidney proximal tubule epithelium, where native human URAT1 is localized in human kidney. Upon hypoxanthine challenge, the blood uric acid (UA) level was elevated in hURAT1-KI mice (251 μmol/L), showing an approximately 37% increase compared to wild-type (WT) mice (183.5 μmol/L). The elevated blood UA level could be alleviated by hURAT1 inhibitor benzbromarone treatment in the hURAT1-KI mice (164.2 μmol/L vs. 251 μmol/L, p < 0.05) whereas no response was observed in WT littermates (168.8 μmol/L vs. 183.5 μmol/L).
CONCLUSION: The hURAT1-KI hyperuricemia mouse model would be valuable for preclinical evaluation of gout treatment with urate-lowering drugs and for studying UA metabolic complexities in humans.}, }
@article {pmid39739718, year = {2024}, author = {Delgado-Nungaray, JA and Figueroa-Yáñez, LJ and Reynaga-Delgado, E and Corona-España, AM and Gonzalez-Reynoso, O}, title = {Unveiling the endogenous CRISPR-Cas system in Pseudomonas aeruginosa PAO1.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0312783}, pmid = {39739718}, issn = {1932-6203}, mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *CRISPR-Cas Systems ; Genome, Bacterial ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Multidrug resistance in Pseudomonas aeruginosa, a high-priority pathogen per the World Health Organization, poses a global threat due to carbapenem resistance and limited antibiotic treatments. Using the bioinformatic tools CRISPRCasFinder, CRISPRCasTyper, CRISPRloci, and CRISPRImmunity, we analyzed the genome of P. aeruginosa PAO1 and revealed an orphan CRISPR system, suggesting it may be a remnant of a type IV system due to the presence of the DinG protein. This system comprises two CRISPR arrays and noteworthy DinG and Cas3 proteins, supporting recent evidence about the association between type IV and I CRISPR systems. Additionally, we demonstrated a co-evolutionary relationship between the orphan CRISPR system in P. aeruginosa PAO1 and the mobile genetic element and prophages identified. One self-targeting spacer was identified, often associated with bacterial evolution and autoimmunity, and no Acr proteins. This research opens avenues for studying how these CRISPR arrays regulate pathogenicity and for developing alternative strategies using its endogenous orphan CRISPR system against carbapenem-resistant P. aeruginosa strains.}, }
@article {pmid39738995, year = {2024}, author = {Argentel-Martínez, L and Peñuelas-Rubio, O and Herrera-Sepúlveda, A and González-Aguilera, J and Sudheer, S and Salim, LM and Lal, S and Pradeep, CK and Ortiz, A and Sansinenea, E and Hathurusinghe, SHK and Shin, JH and Babalola, OO and Azizoglu, U}, title = {Biotechnological advances in plant growth-promoting rhizobacteria for sustainable agriculture.}, journal = {World journal of microbiology & biotechnology}, volume = {41}, number = {1}, pages = {21}, pmid = {39738995}, issn = {1573-0972}, mesh = {*Agriculture/methods ; *Bacteria/genetics/classification/metabolism ; *Biotechnology/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/microbiology/growth & development ; Gene Editing/methods ; *Genetic Engineering/methods ; Microbiota ; Plant Development ; Plant Roots/microbiology/growth & development ; *Rhizosphere ; Soil Microbiology ; }, abstract = {The rhizosphere, the soil zone surrounding plant roots, serves as a reservoir for numerous beneficial microorganisms that enhance plant productivity and crop yield, with substantial potential for application as biofertilizers. These microbes play critical roles in ecological processes such as nutrient recycling, organic matter decomposition, and mineralization. Plant growth-promoting rhizobacteria (PGPR) represent a promising tool for sustainable agriculture, enabling green management of crop health and growth, being eco-friendly alternatives to replace chemical fertilizers and pesticides. In this sense, biotechnological advancements respecting genomics and gene editing have been crucial to develop microbiome engineering which is pivotal in developing microbial consortia to improve crop production. Genome mining, which involves comprehensive analysis of the entire genome sequence data of PGPR, is crucial for identifying genes encoding valuable bacterial enzymes and metabolites. The CRISPR-Cas system, a cutting-edge genome-editing technology, has shown significant promise in beneficial microbial species. Advances in genetic engineering, particularly CRISPR-Cas, have markedly enhanced grain output, plant biomass, resistance to pests, and the sensory and nutritional quality of crops. There has been a great advance about the use of PGPR in important crops; however, there is a need to go further studying synthetic microbial communities, microbiome engineering, and gene editing approaches in field trials. This review focuses on future research directions involving several factors and topics around the use of PGPR putting special emphasis on biotechnological advances.}, }
@article {pmid39738767, year = {2024}, author = {Lei, Z and Cai, H and Yan, Q}, title = {NF2 can mediate the expression of CAMK2A in a tissue specific manner.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {31992}, pmid = {39738767}, issn = {2045-2322}, mesh = {Humans ; *Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism/genetics ; *Gene Expression Regulation, Neoplastic ; Meningioma/genetics/metabolism/pathology ; Neurofibromin 2/metabolism/genetics ; Organ Specificity/genetics ; Meningeal Neoplasms/genetics/metabolism/pathology ; Calcium/metabolism ; }, abstract = {Meningioma is the most prevalent primary intracranial tumor, with approximately half of patients harboring NF2 alteration. The rationale behind the presence of NF2 alteration in meningiomas and its absence in non-nerve system tumors remains elusive. Therefore, meningiomas and several non-nerve system tumor types were analyzed using KEGG analysis and CRISPR/Cas 9 technology to determine the role of NF2 in regulating tissue specificity. Moreover, the different regulatory patterns of Ca[2+] and calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) that play a decisive role in NF2 tissue-specific regulation were deciphered. Our results revealed that NF2 has a positive correlation in CAMK2A expression in both meningiomas and normal nervous system tissues but not in non-nervous system tumors and tissues, implying NF2 tissue-specificity is mediated by CAMK2A-related pathways. Thus, targeting CAMK2A may represent a promising strategy for drug screening and the development of therapeutics for NF2-associated meningiomas and other nervous system tumors.}, }
@article {pmid39738613, year = {2024}, author = {Koshi, D and Sugano, J and Yamasaki, F and Kawauchi, M and Nakazawa, T and Oh, M and Honda, Y}, title = {Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences.}, journal = {Applied microbiology and biotechnology}, volume = {108}, number = {1}, pages = {548}, pmid = {39738613}, issn = {1432-0614}, support = {K-2019-002//Institute for Fermentation, Osaka/ ; 18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; 23KJ1317//Japan Society for the Promotion of Science/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Pleurotus/genetics ; Genome, Fungal/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus/genetics ; Plasmids/genetics ; CRISPR-Associated Protein 9/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG[+] recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs. KEY POINTS: • Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus. • Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences. • Efficient genome editing protocol for safer molecular breeding in mushroom fungus.}, }
@article {pmid39738601, year = {2024}, author = {Esmaeili, M and Smith, DA and Mead, B}, title = {miRNA changes associated with differentiation of human embryonic stem cells into human retinal ganglion cells.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {31895}, pmid = {39738601}, issn = {2045-2322}, support = {5183/5184//Fight for Sight UK/ ; }, mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Retinal Ganglion Cells/metabolism/cytology ; *Cell Differentiation/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Animals ; Rats ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {miRNA, short non-coding RNA, are rapidly emerging as important regulators in cell homeostasis, as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC), whose axons connect the eye to the brain, are central nervous system cells of great interest, yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line, human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC, with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA's role in RGC function, as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration.}, }
@article {pmid39737993, year = {2024}, author = {Yuan, Q and Zeng, H and Daniel, TC and Liu, Q and Yang, Y and Osikpa, EC and Yang, Q and Peddi, A and Abramson, LM and Zhang, B and Xu, Y and Gao, X}, title = {Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10868}, pmid = {39737993}, issn = {2041-1723}, support = {CBET-2143626//National Science Foundation (NSF)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Trans-Activators/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Homeodomain Proteins/genetics/metabolism ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; RNA/genetics ; Genome, Human ; CRISPR-Cas Systems ; Mutation ; RNA Interference ; Genetic Engineering/methods ; }, abstract = {Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2-p65-HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson's disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.}, }
@article {pmid39737984, year = {2024}, author = {Nagamura, R and Kujirai, T and Kato, J and Shuto, Y and Kusakizako, T and Hirano, H and Endo, M and Toki, S and Saika, H and Kurumizaka, H and Nureki, O}, title = {Structural insights into how Cas9 targets nucleosomes.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10744}, pmid = {39737984}, issn = {2041-1723}, support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; JP19am0401005//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121002//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121012//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121009//Japan Agency for Medical Research and Development (AMED)/ ; JP23H05475//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJER1901//MEXT | JST | Exploratory Research for Advanced Technology (ERATO)/ ; }, mesh = {*Nucleosomes/metabolism/ultrastructure ; *Cryoelectron Microscopy ; *DNA/metabolism/chemistry ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *Gene Editing ; Histones/metabolism/chemistry ; CRISPR-Cas Systems ; Chromatin/metabolism/chemistry ; DNA Cleavage ; Models, Molecular ; }, abstract = {The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.}, }
@article {pmid39737975, year = {2024}, author = {Wu, Y and Zhong, A and Sidharta, M and Kim, TW and Ramirez, B and Persily, B and Studer, L and Zhou, T}, title = {Robust and inducible genome editing via an all-in-one prime editor in human pluripotent stem cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10824}, pmid = {39737975}, issn = {2041-1723}, support = {P30 CA008748/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; Genome, Human ; Mutation ; Cell Line ; }, abstract = {Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small insertions or deletions at a specific genomic locus. Here, we systematically compare a panel of prime editing conditions in hPSCs and generate a potent prime editor, "PE-Plus", through co-inhibition of mismatch repair and p53-mediated cellular stress responses. We further establish an inducible prime editing platform in hPSCs by incorporating the PE-Plus into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.}, }
@article {pmid39737904, year = {2024}, author = {Li, D and Zhang, S and Lin, S and Xing, W and Yang, Y and Zhu, F and Su, D and Chen, C and Liu, JG}, title = {Cas12e orthologs evolve variable structural elements to facilitate dsDNA cleavage.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10727}, pmid = {39737904}, issn = {2041-1723}, support = {32150018//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21877069//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22277063//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22061160466//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101195//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022M711848//China Postdoctoral Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/genetics/chemistry ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Gene Editing/methods ; Evolution, Molecular ; Protein Domains ; }, abstract = {Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties. To comprehensively understand the Cas12e family, we identify and characterize six unreported Cas12e members that vary in their CRISPR-locus architectures, PAM preferences, and cleavage efficacies. Interestingly, among all variants, PlmCas12e exhibits the most robust trans-cleavage activity and the lowest salt sensitivity in cis-cleavage. Further structural comparisons reveal that the unique NTSB domain in PlmCas12e is beneficial to DNA unwinding at high salt concentrations, while some NTSB-lacking Cas12e proteins rely on positively charged loops for dsDNA unwinding. These findings demonstrate how divergent evolution of structural elements shapes the nuclease diversity within the Cas12e family, potentially contributing to their adaptations to varying environmental conditions.}, }
@article {pmid39736572, year = {2024}, author = {Stelmach-Wityk, K and Szymonik, K and Grzebelus, E and Kiełkowska, A}, title = {Development of an optimized protocol for protoplast-to-plant regeneration of selected varieties of Brassica oleracea L.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {1279}, pmid = {39736572}, issn = {1471-2229}, mesh = {*Protoplasts ; *Brassica/genetics/physiology/growth & development ; *Regeneration ; Plant Shoots/genetics/growth & development ; Plant Breeding/methods ; Plant Roots/genetics/growth & development/physiology ; }, abstract = {BACKGROUND: Brassica oleracea L. is a key plant in the Brassicaceae family, known for popular vegetables like cabbage, broccoli, kale and collard. Collard (B. oleracea var. viridis) is a non-heading leafy vegetable grown in urban farms and community gardens in the United States and Europe. Improving collard and other Brassica germplasm can benefit from both traditional and new plant breeding technologies (NPBTs), such as CRISPR-Cas mediated transformation. An efficient transformation or protoplast fusion can only be achieved with a robust and reproducible protocol for protoplast-to-plant regeneration. This research focuses on optimizing in vitro culture conditions to enhance cell divisions, microcallus formation, and the regeneration of shoots and roots in four Brassica oleracea varieties, including collard.
RESULTS: The protocol of protoplast release, purification and immobilization was optimized to obtain a suitable number and quality of protoplasts from seven cultivars of B. oleracea. The protoplast isolation efficiency after digestion of young leaves in optimized enzyme solution reached on average 2.5 × 10[6] of cells per gram of fresh weight. Protoplasts were embedded in thin alginate layers and subjected to culture in three different media. Protoplasts of all studied cultivars were viable (88.2%), underwent cell wall resynthesis and re-entered mitotic divisions in the 5th day of culture. After 30 days of culture, protoplast-derived cells of all the tested cultivars formed microcallus. Six cultivars regenerated shoots, although the shoot formation efficiency strongly depended on the genotype and composition of the regeneration medium. The regeneration medium supplemented with 1 mg l[-1] of NAA, 1 mg l[-1] of 2iP, 0.02 mg l[-1] GA3 and with 2% of mannitol showed the highest shoot formation efficiency for five cultivars of B. oleracea.
CONCLUSIONS: The results of this research have led to the development of a robust protoplast-to-plant regeneration protocol for four varieties of B. oleracea that could be exploited as a tool for production of transformants and somatic hybrids. Furthermore, we present the first successful regeneration of protoplast-derived plants of collard, an overlooked but valuable variety of Brassica oleracea.}, }
@article {pmid39735189, year = {2024}, author = {Lu, Z and Liang, M and Li, C and Xu, Y and Weng, S and He, J and Guo, C}, title = {Rapid, sensitive, and visual detection of mandarin fish ranavirus and infectious spleen and kidney necrosis virus using an RPA-CRISPR/Cas12a system.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1495777}, pmid = {39735189}, issn = {1664-302X}, abstract = {Iridoviruses are large cytoplasmic icosahedral viruses that contain dsDNA. Among them, mandarin fish ranavirus (MRV) and infectious spleen and kidney necrosis virus (ISKNV) are particularly notable due to their high contagiousness and pathogenicity. These viruses pose a significant threat to fish aquaculture, resulting in substantial annual economic losses for the fish farming industry. Therefore, the development of novel, rapid virus detection technologies is essential for the prevention and control of ISKNV and MRV diseases. In this study, we developed a rapid, sensitive, and visual detection method for MRV and ISKNV using the recombinase polymerase amplification (RPA)-CRISPR/Cas12a system. This method can detect as low as 1 copy/μL of MRV and 0.1 copy/μL of ISKNV, demonstrating excellent specificity and reproducibility. The detection can be performed at a constant temperature of 37-39°C, eliminating the need for complex equipment. A 30-min RPA amplification followed by a 15-min CRISPR/Cas reaction is sufficient for detecting most samples. For low-concentration samples, extending the CRISPR/Cas reaction time to 60 min improves result visibility. The designed RPA reaction system is capable of performing reverse transcription of RNA, allowing for the detection of mRNA transcribed from the MCP gene of MRV and ISKNV in the sample. Furthermore, two probes were identified that can be observed without the need for excitation light. In conclusion, a field-suitable detection method for ISKNV and MRV has been established, providing a powerful tool for the prompt diagnosis of these aquatic pathogens and aiding in the prevention and control of ISKNV and MRV diseases.}, }
@article {pmid39734219, year = {2024}, author = {Wen, Q and Chen, J and Li, J and Dharmasiddhi, IPW and Yang, M and Xing, J and Liu, Y}, title = {A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440.}, journal = {Microbial cell factories}, volume = {23}, number = {1}, pages = {349}, pmid = {39734219}, issn = {1475-2859}, support = {2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; }, mesh = {*Pseudomonas putida/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; Genome, Bacterial ; }, abstract = {BACKGROUND: Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes.
RESULTS: We discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 [o]C. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system's user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield.
CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system's potential for expanding biotechnological applications.}, }
@article {pmid39730605, year = {2024}, author = {Sassone, F and Estay-Ahumada, C and Roux, MJ and Ciocca, D and Rossolillo, P and Birling, MC and Sparrow, JR and Montenegro, D and Hicks, D}, title = {Interruption of the visual cycle in a novel animal model induces progressive vision loss resembling Stargardts Disease.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30880}, pmid = {39730605}, issn = {2045-2322}, support = {R01 EY012951/EY/NEI NIH HHS/United States ; NEI EY012951//UNADEV/ITMO Aviesan 2018-2021, Fondation de France (Association Berthe Fouassier) and USIAS (DH); and NEI EY012951 (JRS)./ ; }, mesh = {Animals ; *Disease Models, Animal ; *ATP-Binding Cassette Transporters/genetics/metabolism ; *Stargardt Disease ; Humans ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Macular Degeneration/pathology/genetics/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Rats ; Retina/metabolism/pathology/physiopathology ; Tomography, Optical Coherence ; Retinal Degeneration/metabolism/pathology/genetics ; }, abstract = {Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts Disease type 1 (STGD1), the most common form of inherited macular degeneration. STGD1 typically declares early in life and leads to severe visual handicap. Abca4 gene-deletion mouse models of STGD1 accumulate lipofuscin, a hallmark of the disease, but unlike the human disease show no or only moderate structural changes and no functional decline. The human macula is highly enriched in cones, and reasoning that the low cone percentage in mice retinas (< 3%) might compromise faithful modelling of human maculopathies, we performed sub-retinal injections of CRISPR/Cas9-abca4 Adeno-Associated Virus constructs into young Sand Rats (Psammomys obesus), a diurnal rodent containing > 30% cones. Compared to control injections of AAV-abca4-GFP, treated eyes exhibited extensive retinal degeneration by two months. Sanger sequencing of the CRISPR targeted sequence show a clear edition of Abca4 gene. Non-invasive fundus imaging showed widespread photoreceptor loss, confirmed by ocular coherence tomography. Functional recording by single flash and flicker electroretinography showed significant decline in photopic (cone) light responses. Post-mortem real-time PCR, immunohistochemistry and western blotting showed significant decrease of cone-specific (MW cone opsin) but not rod-specific (rhodopsin) markers. Transmission electron microscopy showed large numbers of lipid inclusions in treated but not control retinal pigmented epithelium. Finally, ultra-high performance liquid chromatography analysis of whole P. obesus eyes showed the presence of all-trans retinal-dimer, not detected in rod-rich rat eyes. In conclusion, Abca4 knockout in P. obesus results in a predominantly cone degeneration phenotype, more accurately reflecting the etiology of human STGD1, and should be valuable for characterizing pathogenic pathways and exploring treatment options.}, }
@article {pmid39730601, year = {2024}, author = {Nasrallah, A and Rezvani, HR and Kobaisi, F and Hammoud, A and Rambert, J and Smits, JPH and Sulpice, E and Rachidi, W}, title = {Generation and characterization of CRISPR-Cas9-mediated XPC gene knockout in human skin cells.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30879}, pmid = {39730601}, issn = {2045-2322}, support = {ANR-18-CE17-0017//ANR grant PG2HEAL/ ; ANR-15-IDEX-02//the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02)./ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA-Binding Proteins/genetics/metabolism ; *DNA Repair ; *Xeroderma Pigmentosum/genetics ; *Gene Knockout Techniques ; *Ultraviolet Rays/adverse effects ; Skin/metabolism/pathology/radiation effects ; DNA Damage ; Keratinocytes/metabolism ; Fibroblasts/metabolism/radiation effects ; Melanocytes/metabolism/radiation effects ; }, abstract = {Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.}, }
@article {pmid39730554, year = {2024}, author = {Cheng, Y and Zhang, L and Ke, Y and Dang, X and Miki, D}, title = {Double step screening using endogenous marker improves relative gene targeting efficiency in Arabidopsis.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30791}, pmid = {39730554}, issn = {2045-2322}, support = {20ZR1467000//Shanghai Science and Technology Innovation Plan/ ; G202201355L//Foreign Expert Project/ ; }, mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems ; *Gene Targeting/methods ; Genetic Markers ; Plants, Genetically Modified/genetics ; Arabidopsis Proteins/genetics/metabolism ; Genome, Plant ; Gene Editing/methods ; }, abstract = {Gene targeting (GT) is a powerful tool for manipulating endogenous genomic sequences as intended. However, its efficiency is rather low, especially in seed plants. Numerous attempts have been made to improve the efficiency of GT via the CRISPR/Cas systems in plants, but these have not been sufficiently effective to be used routinely by everyone. Here, we report a surrogate screening method that improves the relative efficiency of CRISPR/Cas9-mediated GT in Arabidopsis. Our findings indicate that simultaneous mutagenesis of the endogenous MAR1 gene, which results in kanamycin resistance, can be employed to efficiently screen for precise and heritable GT events at multiple endogenous sites in the Arabidopsis genome. In this study, we demonstrate that a double-step screening strategy can achieve up to a four-fold increase in the efficiency of GT in Arabidopsis. The principle of this surrogate system has the potential to be widely applied.}, }
@article {pmid39729269, year = {2025}, author = {Qiu, M and Chen, N and Wang, Y}, title = {CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {69-82}, pmid = {39729269}, issn = {1940-6029}, mesh = {*Phytophthora/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Complementation Test ; Plasmids/genetics ; Genetic Vectors/genetics ; }, abstract = {The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.}, }
@article {pmid39729268, year = {2025}, author = {Mendoza, CS and Ah-Fong, AMV and Judelson, HS}, title = {Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {49-67}, pmid = {39729268}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *Phytophthora infestans/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Recombinational DNA Repair ; Genetic Vectors/genetics ; Endodeoxyribonucleases ; }, abstract = {Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.}, }
@article {pmid39727993, year = {2024}, author = {Mascarenhas, MS and Nascimento, FDS and Schittino, LMP and Galinari, LB and Lino, LSM and Ramos, APS and Diniz, LEC and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP}, title = {Construction and Validation of CRISPR/Cas Vectors for Editing the PDS Gene in Banana (Musa spp.).}, journal = {Current issues in molecular biology}, volume = {46}, number = {12}, pages = {14422-14437}, pmid = {39727993}, issn = {1467-3045}, support = {OPP1093845//IITA - Accelerated Breeding of Better Bananas/ ; OPP1093845//Bill and Melinda Gates Foundation - Accelerated Breeding of Better Bananas/ ; }, abstract = {Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors.}, }
@article {pmid39727898, year = {2024}, author = {Zhang, X and Huang, Z and Zhang, Y and Wang, W and Ye, Z and Liang, P and Sun, K and Kang, W and Tang, Q and Yu, X}, title = {Mitigating Antibiotic Resistance: The Utilization of CRISPR Technology in Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727898}, issn = {2079-6374}, support = {2022C02049//the "Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; 2024SNJF044//Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 2023SNJF066//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; }, mesh = {*CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Bacteria ; Biosensing Techniques ; Drug Resistance, Bacterial ; }, abstract = {Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.}, }
@article {pmid39727873, year = {2024}, author = {Lu, Z and Ye, Z and Li, P and Jiang, Y and Han, S and Ma, L}, title = {An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727873}, issn = {2079-6374}, support = {WDZC20200821104802001//Universities Stable Funding Key Projects/ ; 2020YFA0908900//The National Key R&D Program of China/ ; n/a//State Key Laboratory of Chemical Oncogenomics/ ; }, mesh = {*DNA Methylation ; *CRISPR-Cas Systems ; Humans ; *Glycerol ; DNA Restriction Enzymes ; Biosensing Techniques ; Nasopharyngeal Carcinoma/diagnosis/genetics ; Nucleic Acid Amplification Techniques ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed.
METHODS: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity.
RESULTS: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.}, }
@article {pmid39727179, year = {2024}, author = {Lopez, SC and Lee, Y and Zhang, K and Shipman, SL}, title = {SspA is a transcriptional regulator of CRISPR adaptation in E. coli.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1244}, pmid = {39727179}, issn = {1362-4962}, support = {MCB 2137692//National Science Foundation/ ; //Pew Biomedical Scholars Program/ ; //Chan Zuckerberg Biohub - San Francisco/ ; //Berkeley Fellowship for Graduate Study/ ; }, abstract = {The CRISPR integrases Cas1-Cas2 create immunological memories of viral infection by storing phage-derived DNA in CRISPR arrays, a process known as CRISPR adaptation. A number of host factors have been shown to influence adaptation, but the full pathway from infection to a fully integrated, phage-derived sequences in the array remains incomplete. Here, we deploy a new CRISPRi-based screen to identify putative host factors that participate in CRISPR adaptation in the Escherichia coli Type I-E system. Our screen and subsequent mechanistic characterization reveal that SspA, through its role as a global transcriptional regulator of cellular stress, is required for functional CRISPR adaptation. One target of SspA is H-NS, a known repressor of CRISPR interference proteins, but we find that the role of SspA on adaptation is not H-NS-dependent. We propose a new model of CRISPR-Cas defense that includes independent cellular control of adaptation and interference by SspA.}, }
@article {pmid39726635, year = {2024}, author = {Brant, EJ and May, D and Eid, A and Altpeter, F}, title = {Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1505844}, pmid = {39726635}, issn = {2673-3439}, abstract = {Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100-130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.}, }
@article {pmid39726634, year = {2024}, author = {Azeez, SS and Hamad, RS and Hamad, BK and Shekha, MS and Bergsten, P}, title = {Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1509924}, pmid = {39726634}, issn = {2673-3439}, abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.}, }
@article {pmid39726403, year = {2024}, author = {Rasool, HMH and Chen, Q and Gong, X and Zhou, J}, title = {CRISPR/Cas system and its application in the diagnosis of animal infectious diseases.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {38}, number = {24}, pages = {e70252}, pmid = {39726403}, issn = {1530-6860}, support = {2024BEG02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; CAAS-ZDRW202410//ASTIP | The Agricultural Science and Technology Innovation Program/ ; }, mesh = {Animals ; Humans ; *Communicable Diseases/diagnosis/genetics/veterinary ; *CRISPR-Cas Systems ; Molecular Diagnostic Techniques/methods ; Zoonoses/diagnosis/genetics ; }, abstract = {Infectious diseases are a serious threat to the existence of animals and humans' life. In the 21st century, the emergence and re-emergence of several zoonotic and non-zoonotic global pandemic diseases of socio-economic importance has affected billions of humans and animals. The need for expensive equipment and laboratories, non-availability of on-site testing abilities, with time-consuming and low sensitivity and specificity issues of currently available diagnostic techniques to identify these pathogenic micro-organisms on a large scale highlighted the need for developing cheap, portable environment friendly diagnostic methods. In recent years, these issues have been addressed by clustered regularly interspaced palindromic repeats (CRISPR)-based diagnostic platforms that have transformed the molecular diagnostic field due to their outstanding ultra-sensitive nucleic acid detecting capabilities. In this study, we highlight the types, potential of different Cas proteins, and amplification systems. We also illuminate the application of currently available CRISPR integrated setups on the diagnosis of infectious diseases, majorly in food-producing animals (pigs, ruminants, poultry, and aquaculture), domestic pets (dogs and cats), and diseases of zoonotic importance. We conclude the challenges and future perspectives of using these systems to rapidly diagnose and treat other infectious diseases and also develop control strategies to prevent the spread of pathogenic organisms.}, }
@article {pmid39725739, year = {2024}, author = {Pi, N and Xiang, R and Zhu, L and Li, Y and Wu, X}, title = {An HRP-integrated CRISPR/Cas12a biosensor towards chair-side diagnosis for Porphyromonas gingivalis.}, journal = {Biotechnology letters}, volume = {47}, number = {1}, pages = {15}, pmid = {39725739}, issn = {1573-6776}, support = {32060024//National Natural Science Foundation of China/ ; 32000088//National Natural Science Foundation of China/ ; 2022JJ40050//Natural Science Foundation of Hunan Province/ ; }, mesh = {*Porphyromonas gingivalis/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacteroidaceae Infections/diagnosis/microbiology ; Periodontitis/microbiology/diagnosis ; }, abstract = {Rapid diagnostic tools for Porphyromonas gingivalis (Pg), the primary microorganism responsible for the development of periodontitis, particularly those designed for chair-side applications, could provide substantial health benefits to patients. To address this issue, we developed a CRISPR/Cas12a-based rapid Pg detection method. Dual-gRNA and hairpin reporter strategies were employed to enhance CRISPR/Cas12a reaction efficiency. By modifying the hairpin reporter with HRP, the pre-amplification-free HRP-CRISPR/Cas12a reaction was enabled to produce a colorimetric output, amplifying the detection signal. This method achieved high sensitivity (as low as 33 CFU) without the risk of aerosol contamination from pre-amplification. When testing clinical samples, the method showed high consistency with the reference RT-PCR. Furthermore, compared with RT-PCR, this method only requires room temperature operation, is simpler, and has a shorter detection time of about 35 min. In conclusion, the pre-amplification-free HRP-integrated CRISPR/Cas12a detection method requires no complex equipment, making it an ideal, end-user-friendly approach for chair-side Pg detection.}, }
@article {pmid39724853, year = {2024}, author = {Zhao, L and Zhao, Z and Li, N and Wang, X}, title = {The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127457}, doi = {10.1016/j.talanta.2024.127457}, pmid = {39724853}, issn = {1873-3573}, abstract = {Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.}, }
@article {pmid39724343, year = {2025}, author = {Nakajima, T and Kuwasaki, Y and Yamamoto, S and Otabe, T and Sato, M}, title = {A Red Light-Activatable Endogenous Gene Transcription System with Red-CPTS.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {45-55}, pmid = {39724343}, issn = {1940-6029}, mesh = {Animals ; *Light ; Mice ; *CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; *Transcription, Genetic/radiation effects ; Red Light ; }, abstract = {Red light penetrates deep into mammalian tissues and has low phototoxicity. We developed a red light-activatable photoswitch (MagRed) for deep tissue optogenetics. Using MagRed, we developed a red light-activatable endogenous gene transcription system (Red-CPTS) based on CRISPR-Cas9. Here we provide a detailed protocol for endogenous gene activation using Red-CPTS in cultured mammalian cells and living mice in vivo.}, }
@article {pmid39724342, year = {2025}, author = {Renzl, C and Mayer, G}, title = {Optoribogenetic Modulation of Transcription.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {37-44}, pmid = {39724342}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; Transcription, Genetic ; Light ; Transfection/methods ; HEK293 Cells ; Gene Expression Regulation ; Flow Cytometry/methods ; }, abstract = {Light can be used as a precise and reversible trigger for the activation of optogenetic tools with subcellular resolution. The interaction of the photoreceptor PAL and aptamer 53 was integrated into a CRISPR/dCas9 system, which can be applied for light-controlled activation of gene expression. Here, we describe a protocol for in vitro application of light-dependent overexpression using eBFP as a proof of concept. The experiment can be done in 3 days, which is split into cell seeding, transfection, and evaluation by flow cytometry. The method is broadly applicable including the upregulation of endogenous genes.}, }
@article {pmid39722550, year = {2025}, author = {Victor Atoki, A and Aja, PM and Shinkafi, TS and Ondari, EN and Adeniyi, AI and Fasogbon, IV and Dangana, RS and Shehu, UU and Akin-Adewumi, A}, title = {Exploring the versatility of Drosophila melanogaster as a model organism in biomedical research: a comprehensive review.}, journal = {Fly}, volume = {19}, number = {1}, pages = {2420453}, pmid = {39722550}, issn = {1933-6942}, mesh = {Animals ; *Drosophila melanogaster/genetics ; *Disease Models, Animal ; Biomedical Research ; Humans ; CRISPR-Cas Systems ; }, abstract = {Drosophila melanogaster is a highly versatile model organism that has profoundly advanced our understanding of human diseases. With more than 60% of its genes having human homologs, Drosophila provides an invaluable system for modelling a wide range of pathologies, including neurodegenerative disorders, cancer, metabolic diseases, as well as cardiac and muscular conditions. This review highlights key developments in utilizing Drosophila for disease modelling, emphasizing the genetic tools that have transformed research in this field. Technologies such as the GAL4/UAS system, RNA interference (RNAi) and CRISPR-Cas9 have enabled precise genetic manipulation, with CRISPR-Cas9 allowing for the introduction of human disease mutations into orthologous Drosophila genes. These approaches have yielded critical insights into disease mechanisms, identified novel therapeutic targets and facilitated both drug screening and toxicological studies. Articles were selected based on their relevance, impact and contribution to the field, with a particular focus on studies offering innovative perspectives on disease mechanisms or therapeutic strategies. Our findings emphasize the central role of Drosophila in studying complex human diseases, underscoring its genetic similarities to humans and its effectiveness in modelling conditions such as Alzheimer's disease, Parkinson's disease and cancer. This review reaffirms Drosophila's critical role as a model organism, highlighting its potential to drive future research and therapeutic advancements.}, }
@article {pmid39722520, year = {2024}, author = {Deng, K and Li, M and Zhang, H and Deng, Y and Qin, Y and Qin, C}, title = {[Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {12}, pages = {4605-4615}, doi = {10.13345/j.cjb.240307}, pmid = {39722520}, issn = {1872-2075}, mesh = {Animals ; *ADP-Ribosylation Factors/genetics/metabolism ; *Zika Virus Infection/genetics ; Mice ; *Mice, Knockout ; *Zika Virus/genetics ; *Mice, Inbred C57BL ; *CRISPR-Cas Systems ; Viral Load ; Male ; Female ; }, abstract = {The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO[-/+], ARF5KO[-/-], and ARF4KO[-/+]/ARF5KO[-/-] mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%-50.0% but not ARF5 in ARF4KO[-/+] compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO[-/-] mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung, kidney, and testis were detected in ARF4KO[-/+]/ARF5KO[-/-]mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO[-/+] mice, while it showed no significant change in ARF5KO[-/-] or ARF4KO[-/+]/ARF5KO[-/-] mice compared with that in the wild type. Meanwhile, ARF4KO[-/+] mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.}, }
@article {pmid39722468, year = {2024}, author = {Lohani, N and Singh, MB and Bhalla, PL}, title = {Deciphering the Vulnerability of Pollen to Heat Stress for Securing Crop Yields in a Warming Climate.}, journal = {Plant, cell & environment}, volume = {}, number = {}, pages = {}, doi = {10.1111/pce.15315}, pmid = {39722468}, issn = {1365-3040}, abstract = {Climate change is leading to more frequent and severe extreme temperature events, negatively impacting agricultural productivity and threatening global food security. Plant reproduction, the process fundamental to crop yield, is highly susceptible to heatwaves, which disrupt pollen development and ultimately affect seed-set and crop yields. Recent research has increasingly focused on understanding how pollen grains from various crops react to heat stress at the molecular and cellular levels. This surge in interest over the last decade has been driven by advances in genomic technologies, such as single-cell RNA sequencing, which holds significant potential for revealing the underlying regulatory reprogramming triggered by heat stress throughout the various stages of pollen development. This review focuses on how heat stress affects gene regulatory networks, including the heat stress response, the unfolded protein response, and autophagy, and discusses the impact of these changes on various stages of pollen development. It highlights the potential of pollen selection as a key strategy for improving heat tolerance in crops by leveraging the genetic variability among pollen grains. Additionally, genome-wide association studies and population screenings have shed light on the genetic underpinnings of traits in major crops that respond to high temperatures during male reproductive stages. Gene-editing tools like CRISPR/Cas systems could facilitate precise genetic modifications to boost pollen heat resilience. The information covered in this review is valuable for selecting traits and employing molecular genetic approaches to develop heat-tolerant genotypes.}, }
@article {pmid39721357, year = {2024}, author = {Petruškevičiūtė, A and Šimuliūnaitė, U and Polanco, CM and Rojas, B and Kuras, S and Valatkaitė-Rakštienė, B and Norvilas, R and Vijaya, AK and Muñoz, P and Neniškytė, U and Jakubauskas, A and Burokas, A and Nalvarte, I and Inzunza, J and Naumovas, D and Stoškus, M and Griškevičius, L and Baltriukienė, D and Arias, J}, title = {Generation of a genetically encoded voltage indicator MARINA reporter human iPS cell line using Cas9 (VULSCi002-A-2).}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103628}, doi = {10.1016/j.scr.2024.103628}, pmid = {39721357}, issn = {1876-7753}, abstract = {Fluorescent protein-based Genetically Encoded Voltage Indicators (GEVI) offer a remarkable system for high-throughput screening of membrane potential phenotypes. The GEVI MARINA is a derivative from ArcLight, which conversely to ArcLight increases its fluorescence intensity alongside depolarization. Here we created knock-in reporter human iPS cell lines carrying the MARINA reporter using SpCas9 programmable nuclease and characterize a heterozygous clone.}, }
@article {pmid39720725, year = {2024}, author = {Wi, T and Choi, Y and Kim, J and Choi, YS and Pipkin, ME and Choi, J}, title = {Efficient gene deletion of Integrin alpha 4 in primary mouse CD4 T cells using CRISPR RNA pair-mediated fragmentation.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1445341}, pmid = {39720725}, issn = {1664-3224}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Integrin alpha4/genetics/metabolism/immunology ; *Gene Deletion ; CD4-Positive T-Lymphocytes/immunology/metabolism ; Th1 Cells/immunology ; Cell Differentiation/genetics/immunology ; Mice, Inbred C57BL ; Lymphocytic Choriomeningitis/immunology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lymphocytic choriomeningitis virus/immunology ; }, abstract = {The functional specialization of CD4 T lymphocytes into various subtypes, including TH1 and TFH cells, is crucial for effective immune responses. TFH cells facilitate B cell differentiation within germinal centers, while TH1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating TFH and TH1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on TFH and TH1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the TH1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting.}, }
@article {pmid39719706, year = {2025}, author = {Zhang, AN and Gaston, JM and Cárdenas, P and Zhao, S and Gu, X and Alm, EJ}, title = {CRISPR-Cas spacer acquisition is a rare event in human gut microbiome.}, journal = {Cell genomics}, volume = {5}, number = {1}, pages = {100725}, doi = {10.1016/j.xgen.2024.100725}, pmid = {39719706}, issn = {2666-979X}, mesh = {Humans ; *Gastrointestinal Microbiome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal/genetics ; Bacteriophages/genetics ; Bifidobacterium longum/genetics ; Metagenome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; }, abstract = {Host-parasite relationships drive the evolution of both parties. In microbe-phage dynamics, CRISPR functions as an adaptive defense mechanism, updating immunity via spacer acquisition. Here, we investigated these interactions within the human gut microbiome, uncovering low frequencies of spacer acquisition at an average rate of one spacer every ∼2.9 point mutations using isolates' whole genomes and ∼2.7 years using metagenome time series. We identified a highly prevalent CRISPR array in Bifidobacterium longum spreading via horizontal gene transfer (HGT), with six spacers found in various genomic regions in 15 persons from the United States and Europe. These spacers, targeting two prominent Bifidobacterium phages, comprised 76% of spacer occurrence of all spacers targeting these phages in all B. longum populations. This result suggests that HGT of an entire CRISPR-Cas system introduced three times more spacers than local CRISPR-Cas acquisition in B. longum. Overall, our findings identified key ecological and evolutionary factors in prokaryote adaptive immunity.}, }
@article {pmid39719647, year = {2024}, author = {Yan, L and Song, YS and Zhou, J and Zhu, L and Shi, TW and Yu, HQ and Dong, ZQ and Wang, W and Long, T and Liu, HY and Shi, ZY and Li, JG}, title = {Expression of nicastrin, NICD1, and Hes1 in NCSTN knockout mice: implications for hidradenitis suppurativa, Alzheimer's, and liver cancer.}, journal = {European journal of medical research}, volume = {29}, number = {1}, pages = {622}, pmid = {39719647}, issn = {2047-783X}, support = {81773344//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Transcription Factor HES-1/genetics/metabolism ; *Amyloid Precursor Protein Secretases/genetics/metabolism ; *Mice, Knockout ; *Alzheimer Disease/genetics/metabolism ; Mice ; *Hidradenitis Suppurativa/genetics ; Female ; Male ; *Liver Neoplasms/genetics/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; Receptor, Notch1/genetics ; }, abstract = {BACKGROUND: Nicastrin, a subunit of the γ-secretase complex, is encoded by the NCSTN gene and regulates notch signaling, it is involved in the pathogenesis of hidradenitis suppurativa (HS), Alzheimer disease (AD), and liver cancer. However, the animal models for studying HS are relatively scarce.
METHODS: CRISPR/Cas-mediated genetic engineering was used to generate targeted knockout offspring mice (C57BL/6J). Different doses (10 mg/kg, 20 mg/kg, and 30 mg/kg) and injection methods (subcutaneous/intraperitoneal/gavage injection) of tamoxifen were used to induce the construction of NCSTN knockout mice (mice model). The expressions of nicastrin, NICD1, hes1 in skin, brain, and liver tissue in mice model and wild-type (WT) mice were measured by qRT-PCR and IHC.
RESULTS: The construction of mice model was successfully induced by tamoxifen, knockout efficiency was 93%, there was no difference in knockout efficiency among three doses, injection methods, genders (P > 0.05). HS-like lesions appeared on the skin of NCSTN knockout mice after 1 month of treatment with tamoxifen, male mice had a higher number of skin lesions compared to female mice (male vs female = 76.5% vs 41.7%, P = 0.027). Compared with WT mice, the expressions of nicastrin (skin P = 0.0009, brain P = 0.0194, liver P = 0.0066), NICD1 (skin P = 0.0115, brain P = 0.0307, liver P = 0.008), hes1 (skin P = 0.0476, brain P = 0.0143, liver P = 0.0003) in mice model all decreased.
CONCLUSIONS: The NCSTN knockout mouse might be employed as HS animal model; Reducing nicastrin may affect the expression of notch1-hes1 pathway molecules in skin, brain, and liver tissues; low dose (10 mg/kg/d) tamoxifen could be used to induce the deletion of the target gene in mice.}, }
@article {pmid39719558, year = {2024}, author = {Nadeem, I and Han, Z and Xiaoliang, H and Adzraku, SY and Kambey, PA and Kanwore, K and Peipei, M and Adekunle, AO and Adu-Amankwaah, J and Ayanlaja, AA and Zheng, Y and Dianshuai, G and Liu, X and Song, Y}, title = {Doublecortin regulates the mitochondrial-dependent apoptosis in glioma via Rho-A/Net-1/p38-MAPK signaling.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {30}, number = {1}, pages = {272}, pmid = {39719558}, issn = {1528-3658}, support = {82273250//National Natural Science Foundation of China/ ; KYCX22_2868//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; }, mesh = {Humans ; *Glioma/metabolism/pathology/genetics ; *Apoptosis ; *Mitochondria/metabolism ; Animals ; *Doublecortin Domain Proteins ; *Doublecortin Protein ; Cell Line, Tumor ; *p38 Mitogen-Activated Protein Kinases/metabolism ; *Microtubule-Associated Proteins/metabolism/genetics ; Mice ; *Neuropeptides/metabolism/genetics ; Cell Proliferation ; Brain Neoplasms/metabolism/pathology/genetics ; Temozolomide/pharmacology ; Signal Transduction ; MAP Kinase Signaling System ; Reactive Oxygen Species/metabolism ; }, abstract = {Doublecortin (DCX) is a microtubule-associated protein known to be a key regulator of neuronal migration and differentiation during brain development. However, the role of DCX, particularly in regulating the survival and growth of glioma cells, remains unclear. In this study, we utilized CRISPR/Cas9 technology to knock down DCX in the human glioma cell line (U251). DCX depletion suppressed cell proliferation and enhanced the pro-apoptotic effects of temozolomide (TMZ) and γ-radiation treatment. DCX knockdown led to the translocation of Bax to the mitochondria and mitochondria dysfunction. Furthermore, DCX deficiency-induced apoptosis took place along with the generation of reactive oxygen species (ROS), which is crucial in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, the transcriptional inhibition of DCX downregulated Rho-A, Net-1, and activated p38-MAPK cue, critical for cell survival and proliferation. Subsequent treatment with TMZ and γ-radiation further increased p38-MAPK activity through the decreased expression of Rho-A/Net-1, resulting in a significant reduction in glioma cell migration and invasion. Additionally, intracranial xenograft tumors of DCX-modified U251 cells in nude mice demonstrated inhibited tumor growth. Tumor sections treated with TMZ and γ-radiation exhibited a higher number of TUNEL-positive cells compared to the control group, indicating increased apoptosis. Our finding suggests that DCX depletion reduces glioma cell proliferation and promotes mitochondria-dependent apoptosis by enhancing the chemo and radiotherapy response. Targeting DCX represents a potential therapeutic target for glioma treatment.}, }
@article {pmid39718242, year = {2024}, author = {Dong, J and Hou, C and Deng, L and Gu, T and Zhu, S and Hou, J and Huo, D}, title = {CRISPR/Cas12a-Powered Electrochemical Platform for Dual-miRNA Detection via an AND Logic Circuit.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c06256}, pmid = {39718242}, issn = {1520-6882}, abstract = {The CRISPR/Cas technology shows great potential in molecular detection and diagnostics. However, it is still challenging to detect multiple targets simultaneously using the CRISPR-Cas system. Herein, we ingeniously leverage the synergistic effect of two short single-stranded DNA activators to construct a CRISPR/Cas12a-driven electrochemical sensing platform based on an AND logic circuit ("AND" LC-CRISPR) for the simultaneous detection of dual miRNAs. Specifically, the exponential amplification reaction products triggered by the dual-specific miRNAs are designed as binary inputs to bind with Cas12a/crRNA, forming an AND logic circuit and activating the trans-cleavage ability of the CRISPR-Cas12a system. Subsequently, the hairpin probe biogate on the surface of the functionalized electrochemical signal probe (MB@HP-Fe-MOF) is cleaved by activated Cas12a, leading to the release of the encapsulated electroactive signal molecule methylene blue, thereby generating a strong electrochemical signal. As a result, this "AND" LC-CRISPR sensing platform, requiring only a single crRNA assembled with Cas12a, achieves simultaneous detection of miRNA-155 and miRNA-21 at concentrations as low as 3.2 fM. Moreover, the platform allows easy adjustment of the AND logic circuit inputs according to different detection targets, allowing it to be easily expanded for the analysis and diagnosis of other multibiomarkers. This approach demonstrates promising potential for future applications in intelligent diagnostic medicine.}, }
@article {pmid39717575, year = {2024}, author = {Ahmad, Z and Niyazi, S and Firdoos, A and Wang, C and Manzoor, MA and Ramakrishnan, M and Upadhyay, A and Ding, Y}, title = {Enhancing plant resilience: Nanotech solutions for sustainable agriculture.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40735}, pmid = {39717575}, issn = {2405-8440}, abstract = {The global population growth is driving up the demand for agricultural products, while traditional farming methods like those from the Green Revolution are becoming unsustainable due to climate change. To address these challenges and ensure agricultural sustainability, innovative techniques, such as nanotechnology, are essential to meet rising food demands and enhance agricultural sustainability. Nanotechnology, which promotes a more sustainable and resilient agricultural system while enhancing food security, is a key catalyst for the Agri-tech revolution. This review offers a progressive analysis of nanotechnology's role in managing plant stress. It explores how precision agriculture, particularly via nanosensors, is enhancing our comprehension of plant stress conditions. The integration of nanotechnology with genetic engineering methods, notably CRISPR-Cas technology, is also examined. Furthermore, the review considers the potential toxicological effects of nanoparticles (NPs) on both the environment and plants. Our review has the potential to make a significant impact on human food security by enhancing food production and availability while promoting sustainable agricultural practices. By tackling these challenges, we can contribute to a more reliable and sustainable food supply for the global population.}, }
@article {pmid39716183, year = {2024}, author = {Cohen, S and Bergman, S and Lynn, N and Tuller, T}, title = {A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.}, journal = {Genome medicine}, volume = {16}, number = {1}, pages = {152}, pmid = {39716183}, issn = {1756-994X}, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Software ; Computational Biology/methods ; Gene Editing/methods ; Gene Knockout Techniques ; Computer Simulation ; Models, Genetic ; Gene Expression ; Gene Expression Regulation ; }, abstract = {BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking.
RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models.
CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition . The source code is available at https://github.com/shaicoh3n/EXPosition .}, }
@article {pmid39715238, year = {2024}, author = {Qiu, M and Tian, Y and Wang, H and Yang, J and Qu, B and Jiang, Y and Zhao, Q and Zhang, X and Man, C}, title = {CRISPR/Cas System Meets CLICK-17 DNAzyme: A Click Chemistry-Based Fluorescence Biosensing Platform Designed for Highly Sensitive Detection of Salmonella.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c05316}, pmid = {39715238}, issn = {1520-6882}, abstract = {Salmonella is one of the most dangerous and contagious foodborne pathogens, posing a significant threat to public health and food safety. In this study, we developed a click chemistry-based fluorescence biosensing platform for highly sensitive detection of Salmonella enterica (S. enterica) by integrating the trans-cleavage activity of CRISPR/Cas12a with the CLICK17-mediated copper(II)-dependent azide-alkyne cycloaddition (Cu(II)AAC) click reaction. Herein, CLICK-17 can provide binding sites for Cu ions and high redox stability for one or much catalytically vital Cu[+] within its active sites, which facilitate the click reaction. With the existence of only Cu[2+], CLICK17 still can catalyze the click reaction between 3-butyn-1-ol and 3-azido-7-hydroxycoumarin to produce a fluorescence signal. By integrating the recombinase polymerase amplification (RPA), specific recognition, and trans-cleavage ability of the CRISPR/Cas12a system and the CLICK17-catalyzed Cu(II)AAC click reaction, the established biosensor obtained high detection sensitivity. This CLICK17-assisted CRISPR/Cas12a fluorescence biosensor was used for the detection of S. enterica with a limit of detection (LOD) as low as 1 cfu/mL in a wide linear detection range of 6 × 10[1]-6 × 10[7] cfu/mL. Moreover, the developed biosensor exhibited high specificity and anti-interference capability and had a recovery of 93%-104% in detection of S. enterica in spiked milk, infant formula, orange juice, and meat samples. This study provides a promising CRISPR/Cas12a-based fluorescence biosensor for the detection of foodborne pathogens.}, }
@article {pmid39714464, year = {2024}, author = {Böck, D and Wilhelm, M and Mumenthaler, J and Carpanese, DF and Kulcsár, PI and d'Aquin, S and Cremonesi, A and Rassi, A and Häberle, J and Patriarchi, T and Schwank, G}, title = {Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714464}, issn = {2050-084X}, support = {310030_185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_196455/SNSF_/Swiss National Science Foundation/Switzerland ; FN20-0000000203//Novartis Foundation for Medical-Biological Research/ ; 196287/SNSF_/Swiss National Science Foundation/Switzerland ; 891959/ERC_/European Research Council/International ; }, mesh = {Animals ; *Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Mice ; *Disease Models, Animal ; *Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; *Parkinson Disease/genetics/metabolism ; *Astrocytes/metabolism ; Dopaminergic Neurons/metabolism ; Gene Editing ; Male ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/metabolism/genetics ; Mice, Inbred C57BL ; }, abstract = {Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.}, }
@article {pmid39714446, year = {2024}, author = {Petazzi, P and Ventura, T and Luongo, FP and McClafferty, H and May, A and Taylor, HA and Shipston, MJ and Romanò, N and Forrester, LM and Menendez, P and Fidanza, A}, title = {A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714446}, issn = {2050-084X}, support = {S002219/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EHA RAG 2021//European Hematology Association/ ; Research Global Award//American Society for Hematology/ ; Precision Medicine PhD scholarship/MRC_/Medical Research Council/United Kingdom ; Tissue Repair PhD studentship//College of Medicine and Veterinary Medicine, University of Edinburgh/ ; Traineeship Program 2016/2017//Erasmus +/ ; PERIS program//Catalan Government/ ; RTC-2018-4603-1//MINECO/ ; }, mesh = {*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics ; Humans ; *Cell Differentiation ; *Hematopoietic Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Pluripotent Stem Cells/metabolism ; }, abstract = {A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.}, }
@article {pmid39714100, year = {2024}, author = {Cui, Q and Zhang, Z and Qin, L and Teng, Z and Wang, Z and Wu, W and Fan, L and Su, J and Hao, Y and Qin, J and Zhang, L and Wang, Q and Zhuang, Y and Zheng, H and Zhang, S and Geng, X and Zhu, L and Chen, Y and Lu, B and Li, Y and Zhu, X}, title = {Interleukin-37 promotes wound healing in diabetic mice by inhibiting the MAPK/NLRP3 pathway.}, journal = {Journal of diabetes investigation}, volume = {}, number = {}, pages = {}, doi = {10.1111/jdi.14389}, pmid = {39714100}, issn = {2040-1124}, support = {3030294002//Multidisciplinary Diagnosis and Treatment (MDT) Construction Project of Diabetic Foot/ ; 82400978//National Natural Science Foundation of China/ ; 82000790//National Natural Science Foundation of China/ ; 82200949//National Natural Science Foundation of China/ ; DGF501069/015//Multidisciplinary Diagnosis and Treatment (MDT) demonstration project in research hospital, Shanghai Medical College, Fudan University/ ; }, abstract = {AIMS/INTRODUCTION: Diabetic foot ulcer (DFU) is a prevalent complication of diabetes characterized by heightened inflammation and impaired wound-healing processes. Interleukin-37 (IL-37) is a natural suppressor of innate inflammation. Here, we aim to investigate the potential of IL-37 in enhancing the healing process of diabetic wounds.
MATERIALS AND METHODS: The skin samples of DFU and non-diabetic patients during foot and ankle orthopedic surgery were collected. The IL-37 transgenic mice (IL-37Tg) were created using CRISPR/Cas-mediated genome engineering. Mice were administered streptozotocin (STZ, 150 mg/kg) to induce a diabetic model. After 4 weeks, an equidistant full-thickness excisional wound measuring 8 mm was created on the central back of each mouse and allowed to heal naturally. Body weight and blood glucose levels were measured weekly. The wound area was measured, and skin samples were collected on Day 10 for further Quantitative polymerase chain reaction (qPCR) and WB detection and RNA sequencing analysis.
RESULTS: The proinflammation cytokines such as TNF-α and IL-1β and the MAPK signaling pathway were significantly increased in the wound margin of DFU patients. Compared with diabetic mice, diabetic IL-37Tg mice showed a significantly accelerated healing process. The enriched signaling pathways in RNA sequencing included cytokine-cytokine receptor interaction, TNF signaling pathway, and NOD-like receptor signaling pathway. Through QPCR and WB detection, we found that IL-37 could inhibit the activated MAPK and NOD-like signaling pathway, reducing TNF-α, IL-1β, and NLRP3 expression in the diabetic wound.
CONCLUSIONS: IL-37 promotes skin wound healing in diabetic mice, providing a new possible target for treating diabetic wounds.}, }
@article {pmid39713824, year = {2024}, author = {Karesh, WB}, title = {Shifting from wildlife disease threats to wildlife health.}, journal = {Revue scientifique et technique (International Office of Epizootics)}, volume = {Special Edition}, number = {}, pages = {141-144}, doi = {10.20506/rst.SE.3568}, pmid = {39713824}, issn = {0253-1933}, mesh = {Animals ; *Animals, Wild ; Humans ; Zoonoses/prevention & control ; Conservation of Natural Resources ; Animal Diseases/prevention & control/epidemiology ; }, abstract = {The evolution of wildlife disease management and surveillance, as documented in the World Organisation for Animal Health's Scientific and Technical Review, reflects a deepening understanding of the links between wildlife health, ecosystem integrity and human well-being. Early work, beginning with the World Assembly of Delegates in 1954, primarily focused on diseases like rabies. This focus expanded over time to include broader concerns such as the impacts of climate change, habitat loss and increased human-wildlife interactions on wildlife health. By the late 20th century, the emphasis had shifted towards improved practices for wildlife disease control and the development of advanced diagnostic methods and vaccines. Articles in the Review highlight the growing complexity of wildlife diseases and the need for holistic management strategies. The adoption in recent years of cutting-edge technologies like CRISPR-Cas systems and metagenomics points to a future of more proactive and integrated approaches to wildlife disease management. There is still a need to address not just the consequences of wildlife diseases but also their anthropogenic drivers. The latest perspectives advocate for nature-based solutions, expanded partnerships and systems-level thinking to effectively tackle 21st-century challenges in wildlife and biodiversity conservation.}, }
@article {pmid39712894, year = {2024}, author = {Wang, X and Yang, R and Tang, T and Zhou, Y and Chen, H and Jiang, Y and Zhang, S and Qin, S and Wang, M and Wang, C}, title = {One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1503356}, pmid = {39712894}, issn = {1664-302X}, abstract = {Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.}, }
@article {pmid39710679, year = {2024}, author = {Jia, X and Yuan, B and Wang, W and Wang, K and Ling, D and Wei, M and Hu, Y and Guo, W and Chen, Z and Du, L and Jin, Y}, title = {Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens.}, journal = {Journal of nanobiotechnology}, volume = {22}, number = {1}, pages = {787}, pmid = {39710679}, issn = {1477-3155}, support = {7232261//Beijing Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *Pseudomonas aeruginosa/drug effects/genetics ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Acinetobacter baumannii/drug effects/genetics ; Animals ; *Cations/chemistry ; Mice ; Humans ; Pseudomonas Infections/drug therapy ; Anti-Bacterial Agents/pharmacology/chemistry ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Acinetobacter Infections/drug therapy ; COVID-19 ; Female ; Mice, Inbred BALB C ; Biomimetics/methods ; }, abstract = {In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.}, }
@article {pmid39709563, year = {2024}, author = {Shepelev, MV and Komkov, DS and Golubev, DS and Borovikova, SE and Mazurov, DV and Kruglova, NA}, title = {[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {590-600}, pmid = {39709563}, issn = {0026-8984}, mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CXCR4/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Plasmids/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics/metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; HIV-1/genetics/metabolism ; }, abstract = {To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.}, }
@article {pmid39709562, year = {2024}, author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {575-589}, pmid = {39709562}, issn = {0026-8984}, mesh = {*Receptors, CXCR4/genetics/metabolism ; Humans ; *Gene Knock-In Techniques ; *HIV-1/genetics/drug effects ; HIV Fusion Inhibitors/pharmacology ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair/drug effects/genetics ; Gene Editing/methods ; Nuclear Localization Signals/genetics ; Plasmids/genetics/metabolism ; T-Lymphocytes/metabolism/drug effects ; NF-kappa B/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; HIV Infections/genetics/virology/drug therapy ; }, abstract = {The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.}, }
@article {pmid39709560, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {525-548}, pmid = {39709560}, issn = {0026-8984}, mesh = {Humans ; Animals ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Cycle/genetics ; Homologous Recombination ; }, abstract = {The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.}, }
@article {pmid39709559, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[Current Knowledge of Base Editing and Prime Editing].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {508-524}, pmid = {39709559}, issn = {0026-8984}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Animals ; Genetic Engineering/methods ; DNA/genetics ; }, abstract = {Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.}, }
@article {pmid39709015, year = {2025}, author = {Bigot, S and Ouameur, AA and Roy, G and Fakhfakh, R and Ritt, JF and Légaré, D and Ouellette, M}, title = {Studies of the FBT family transporters in Leishmania infantum by gene deletion and protein localization.}, journal = {Experimental parasitology}, volume = {268}, number = {}, pages = {108880}, doi = {10.1016/j.exppara.2024.108880}, pmid = {39709015}, issn = {1090-2449}, mesh = {*Leishmania infantum/genetics/drug effects/metabolism ; *Gene Deletion ; Cell Membrane/metabolism ; Membrane Transport Proteins/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Methotrexate/pharmacology ; Folic Acid Transporters/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {The protozoan parasite Leishmania has a large family of major facilitator membrane proteins part of the Folate Biopterin Transporter (FBT) family. The chromosome 10 of Leishmania has a cluster of 7 FBT genes including the S-Adenosyl methionine (AdoMet) transporter and the functionally characterized folate transporters FT1 and FT5. Six of the 7 FBT proteins coded by this locus are located at the plasma membrane as determined by gene fusions with the green fluorescent protein. We deleted the whole locus of 7 genes (>30 kb) using CRISPR-Cas9 genome editing as a first step in studying the potential function of the four uncharacterized FBT genes from the locus. This knock out strain was viable, highly resistant to sinefungin (an AdoMet analogue) and to methotrexate (a folate analogue) but not to allopurinol, pentamidine or 5-fluorouracil. We similarly studied another FBT family member whose gene is encoded on chromosome 19. The protein was also located at the plasma membrane and its gene was dispensable for growth and not associated to any of the drug tested. Our work has indicated that large diploid deletion is achievable in Leishmania and the cell lines produced here will serve to better understand the function and putative substrates of these FBT proteins yet to be characterized.}, }
@article {pmid39708170, year = {2024}, author = {Feng, R and Mao, K and Zhang, H and Zhu, H and Du, W and Yang, Z and Wang, S}, title = {Portable microfluidic devices for monitoring antibiotic resistance genes in wastewater.}, journal = {Mikrochimica acta}, volume = {192}, number = {1}, pages = {19}, pmid = {39708170}, issn = {1436-5073}, support = {42377456//the National Natural Science Foundation of China/ ; 2023415//the Youth Innovation Promotion Association CAS/ ; CAS-ANSO-FS-2024-34//CAS-ANSO Fellowship/ ; Qiankehe Jichu-ZK [2022] Yiban 565, Qiankehe Platform Talents-GCC [2023] 046//Guizhou Provincial Science and Technology Projects/ ; }, mesh = {*Wastewater/microbiology ; *Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques/methods ; Drug Resistance, Microbial/genetics ; Genes, Bacterial ; CRISPR-Cas Systems/genetics ; Anti-Bacterial Agents ; Microfluidic Analytical Techniques/instrumentation/methods ; Drug Resistance, Bacterial/genetics ; }, abstract = {Antibiotic resistance genes (ARGs) pose serious threats to environmental and public health, and monitoring ARGs in wastewater is a growing need because wastewater is an important source. Microfluidic devices can integrate basic functional units involved in sample assays on a small chip, through the precise control and manipulation of micro/nanofluids in micro/nanoscale spaces, demonstrating the great potential of ARGs detection in wastewater. Here, we (1) summarize the state of the art in microfluidics for recognizing ARGs, (2) determine the strengths and weaknesses of portable microfluidic chips, and (3) assess the potential of portable microfluidic chips to detect ARGs in wastewater. Isothermal nucleic acid amplification and CRISPR/Cas are two commonly used identification elements for the microfluidic detection of ARGs. The former has better sensitivity due to amplification, but false positives due to inappropriate primer design and contamination; the latter has better specificity. The combination of the two can achieve complementarity to a certain extent. Compared with traditional microfluidic chips, low-cost and biocompatible paper-based microfluidics is a very attractive test for ARGs, whose fluid flow in paper does not require external force, but it is weaker in terms of repeatability and high-throughput detection. Due to that only a handful of portable microfluidics detect ARGs in wastewater, fabricating high-throughput microfluidic chips, developing and optimizing recognition techniques for the highly selective and sensitive identification and quantification of a wide range of ARGs in complex wastewater matrices are needed.}, }
@article {pmid39707688, year = {2024}, author = {Garay-Novillo, JN and Ruiz-Masó, JÁ and Del Solar, G and Barra, JL}, title = {Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.}, journal = {Microbial biotechnology}, volume = {17}, number = {12}, pages = {e70060}, pmid = {39707688}, issn = {1751-7915}, support = {CSIC 2022AEP028//Consejo Superior de Investigaciones Científicas/ ; 2023/ResMi-00000018//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba and Ministerio de Ciencia y Tecnología - Córdoba/ ; PICT Start-Up 2018-00811//Agencia Nacional de Promoción Científica y Tecnológica/ ; CONSOLIDAR RESOL-2023-258-E-UNC-SECYT#ACTIP//Secretaría de Ciencia y Tecnología - Universidad Nacional de Córdoba/ ; MHE200027-EMHE-CSIC-2016-program//Consejo Superior de Investigaciones Científicas/ ; }, mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Lactococcus/genetics ; Hydrogen-Ion Concentration ; }, abstract = {In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.}, }
@article {pmid39707395, year = {2024}, author = {Xu, W and Zhang, S and Qin, H and Yao, K}, title = {From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1133}, pmid = {39707395}, issn = {1479-5876}, support = {82471107//National Natural Science Foundation of China/ ; 31970930//National Natural Science Foundation of China/ ; 2020CFA069//Natural Science Foundation of Hubei Province/ ; 2018CFB434//Natural Science Foundation of Hubei Province/ ; }, mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Animals ; Genetic Therapy/methods ; }, abstract = {CRISPR-based gene editing technology theoretically allows for precise manipulation of any genetic target within living cells, achieving the desired sequence modifications. This revolutionary advancement has fundamentally transformed the field of biomedicine, offering immense clinical potential for treating and correcting genetic disorders. In the treatment of most genetic diseases, precise genome editing that avoids the generation of mixed editing byproducts is considered the ideal approach. This article reviews the current progress of base editors and prime editors, elaborating on specific examples of their applications in the therapeutic field, and highlights opportunities for improvement. Furthermore, we discuss the specific performance of these technologies in terms of safety and efficacy in clinical applications, and analyze the latest advancements and potential directions that could influence the future development of genome editing technologies. Our goal is to outline the clinical relevance of this rapidly evolving scientific field and preview a roadmap for successful DNA base editing therapies for the treatment of hereditary or idiopathic diseases.}, }
@article {pmid39706269, year = {2024}, author = {Li, X and Wei, Y and Wang, SY and Wang, SG and Xia, PF}, title = {One-for-all gene inactivation via PAM-independent base editing in bacteria.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {108113}, doi = {10.1016/j.jbc.2024.108113}, pmid = {39706269}, issn = {1083-351X}, abstract = {Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interest (GOIs) in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a PAM-independent base editor. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.}, }
@article {pmid39706164, year = {2024}, author = {Cenik, BK and Aoi, Y and Iwanaszko, M and Howard, BC and Morgan, MA and Andersen, GD and Bartom, ET and Shilatifard, A}, title = {TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.}, journal = {Molecular cell}, volume = {84}, number = {24}, pages = {4929-4944.e8}, doi = {10.1016/j.molcel.2024.11.007}, pmid = {39706164}, issn = {1097-4164}, mesh = {Humans ; *Promoter Regions, Genetic ; *Transcription Factors/metabolism/genetics ; *Chromatin/metabolism/genetics ; HEK293 Cells ; RNA Polymerase II/metabolism/genetics ; Protein Binding ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Gene Expression Regulation ; Heat-Shock Response/genetics ; Protein Interaction Maps ; Bromodomain Containing Proteins ; }, abstract = {Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.}, }
@article {pmid39705367, year = {2024}, author = {Su, Z and Zhang, W and Shi, Y and Cui, T and Xu, Y and Yang, R and Huang, M and Zhou, C and Zhang, H and Lu, T and Qu, J and He, ZG and Gan, J and Feng, Y}, title = {A bacterial methyltransferase that initiates biotin synthesis, an attractive anti-ESKAPE druggable pathway.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadp3954}, pmid = {39705367}, issn = {2375-2548}, mesh = {*Biotin/biosynthesis ; *Methyltransferases/metabolism/chemistry ; *Acinetobacter baumannii/enzymology ; *Klebsiella pneumoniae/enzymology ; Bacterial Proteins/metabolism/genetics/chemistry ; Adenosine/analogs & derivatives/metabolism ; Anti-Bacterial Agents/pharmacology ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; }, abstract = {The covalently attached cofactor biotin plays pivotal roles in central metabolism. The top-priority ESKAPE-type pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, constitute a public health challenge of global concern. Despite the fact that the late step of biotin synthesis is a validated anti-ESKAPE drug target, the primary stage remains fragmentarily understood. We report the functional definition of two BioC isoenzymes (AbBioC for A. baumannii and KpBioC for K. pneumoniae) that act as malonyl-ACP methyltransferase and initiate biotin synthesis. The physiological requirement of biotin is diverse within ESKAPE pathogens. CRISPR-Cas9-based inactivation of bioC rendered A. baumannii and K. pneumoniae biotin auxotrophic. The availability of soluble AbBioC enabled the in vitro reconstitution of DTB/biotin synthesis. We solved two crystal structures of AbBioC bound to SAM cofactor (2.54 angstroms) and sinefungin (SIN) inhibitor (1.72 angstroms). Structural and functional study provided molecular basis for SIN inhibition of BioC. We demonstrated that BioC methyltransferase plays dual roles in K. pneumoniae infection and A. baumannii colistin resistance.}, }
@article {pmid39705306, year = {2024}, author = {Hou, J and Guo, P and Wang, J and Han, D and Tan, W}, title = {Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2414793121}, pmid = {39705306}, issn = {1091-6490}, mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Biosensing Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Nucleic Acid Conformation ; RNA/chemistry ; Fluorescent Dyes/chemistry ; }, abstract = {Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.}, }
@article {pmid39704190, year = {2024}, author = {Fan, Z and Xu, L and Cao, Y and Liu, T and Tian, Y and Pan, Z and Mo, Y and Wang, X and Zhu, X and Gao, Y and Zhang, X and Pan, CQ and Wang, L and Ren, F}, title = {One-Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point-of-Care Testing.}, journal = {Journal of medical virology}, volume = {96}, number = {12}, pages = {e70115}, pmid = {39704190}, issn = {1096-9071}, support = {//This study was supported by the National Natural Science Foundation of China (82002243, 82100653), Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035), Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY23), Beijing Hospitals Authority Youth Programme (QML20201702), Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration (DFL20221503), Beijing Natural Science Foundation-Changping Innovation Joint Fund (L234046), Training Fund for Open Projects at Clinical Institutes and Departments of Capital Medical University (CCMU2023ZKYXZ003), High-Level Public Health Technical Talents Project of Beijing (Subject Leaders-02-13, xuekegugan-03-48)./ ; }, mesh = {*Hepatitis E virus/genetics/isolation & purification ; *Point-of-Care Testing ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics ; Humans ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Animals ; Limit of Detection ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy-to-use detection. This study aimed to develop a One-Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point-of-care testing (POCT) in resource-limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription recombinase-aided amplification (RT-RAA) was applied to a One-Pot assay device. Additionally, a large cohort of HEV-infected patient (154) and animal (104) specimens was utilized for validation. The RT-PCR/RT-RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV-1, HEV-3, and HEV-4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT-PCR + CRISPR and RT-RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT-qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One-Pot device were integrated to construct a One-Pot assay with an LoD of 10[2] copies/μL. Despite slight decreases in sensitivity, the One-Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One-Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.}, }
@article {pmid39703747, year = {2024}, author = {Fallah, T and Shafiei, M}, title = {Comprehensive Analysis of CRISPR-Cas Systems and Their Influence on Antibiotic Resistance in Salmonella enterica Strains.}, journal = {Bioinformatics and biology insights}, volume = {18}, number = {}, pages = {11779322241307984}, pmid = {39703747}, issn = {1177-9322}, abstract = {Salmonella enterica is a gram-negative bacterium that demonstrates a remarkable ability to acquire antibiotic resistance genes (ARGs). The role of the CRISPR-Cas system in influencing antibiotic resistance in S. enterica is still under investigation. This study explores the distribution and impact of CRISPR-Cas systems on antibiotic resistance by analyzing 316 S. enterica genomes. We conducted sequence alignments, phylogenetic analyses, and conservation studies on Cas genes, direct repeats (DRs), and leader sequences. Promoter predictions and RNA secondary structure analyses were also performed. ARGs were identified, and their correlation with Cas gene clusters was evaluated. Our findings revealed that 82.33% of strains possess complete CRISPR-Cas systems, while 17.66% have orphan CRISPRs. We identified 290 distinct DRs, most of which formed stable stem-loop structures, although no promoter regions were detected within the leader sequences. Most spacers were chromosome-targeting, with a smaller proportion homologous to phages and plasmids. Importantly, strains with complete CRISPR-Cas systems showed a higher incidence of ARGs compared with those with orphan or no CRISPR systems. Specifically, the incidence of ARGs was 54.3% higher in strains with complete CRISPR-Cas systems than in strains without CRISPR-Cas systems, and 15.1% higher than in strains with orphan CRISPRs. Spearman's correlation analysis confirmed a statistically significant but weak correlation between the presence of Cas genes and the frequency of ARGs (P-value = 3.892e-06). These results suggest that CRISPR-Cas systems may play a role in the acquisition of ARGs, potentially through mutations under antibiotic pressure. Future studies should investigate mutations, particularly in Cas3-the signature protein of type I CRISPR-Cas systems. In addition, experimental validation, such as culturing S. enterica strains with complete CRISPR-Cas systems under different antibiotic conditions, followed by sequencing to assess the uptake or absence of newly acquired ARGs, would help clarify the potential role of CRISPR-Cas systems in bacterial adaptation to antimicrobial pressures.}, }
@article {pmid39702810, year = {2024}, author = {Bhagat, M and Kamal, R and Sharma, J and Kaur, K and Sharma, A and Thakur, GS and Bhatia, R and Awasthi, A}, title = {Gene Therapy: Towards a New Era of Medicine.}, journal = {AAPS PharmSciTech}, volume = {26}, number = {1}, pages = {17}, pmid = {39702810}, issn = {1530-9932}, mesh = {Humans ; *Genetic Therapy/methods/trends ; Animals ; *Genetic Vectors ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; }, abstract = {Over the past years, many significant advances have been made in the field of gene therapy and shown promising results in clinical trials conducted. Gene therapy aims at modifying or replacing a defective, inefficient, or nonfunctional gene with a healthy, functional gene by administration of genome material into the cell to cure genetic diseases. Various methods have been devised to do this by using several viral and non-viral vectors which are either administered by in vivo or ex vivo technique. Viral vectors are best suitable for this therapy due to their potential to invade cells and deliver their genetic material whereas non-viral vectors are less efficient than viral vectors but possess some advantages such as less immunogenic response and large gene carrying capacity. Recent advances in biotechnology such as CRISPR-Cas9 mediated genome engineering and Cancer treatment with Chimeric antigen receptor (CAR) T-cell therapy are addressed in this review. This review article also delves into some recent research studies, gene therapy trials, and its applications, laying out future hopes for gene therapy in the treatment of various diseases namely haemophilia, Muscular dystrophy, SCID, Sickle cell disease, Familial Hypercholesterolemia, Cystic Fibrosis. Additionally, it also includes various nanoformulations and clinical trial data related to gene therapy.}, }
@article {pmid39702692, year = {2024}, author = {Du, W and Meister, LL and van Grinsven, T and Branco Dos Santos, F}, title = {Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.}, journal = {Biotechnology and bioengineering}, volume = {}, number = {}, pages = {}, doi = {10.1002/bit.28910}, pmid = {39702692}, issn = {1097-0290}, abstract = {Cyanobacteria have been genetically modified to convert CO2 into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.}, }
@article {pmid39702666, year = {2024}, author = {Kou, Z and Wang, S and Luo, X and Xu, J and Tomberlin, JK and Huang, Y}, title = {Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens.}, journal = {Communications biology}, volume = {7}, number = {1}, pages = {1652}, pmid = {39702666}, issn = {2399-3642}, support = {32021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100381//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; *Diptera/genetics ; Female ; Male ; }, abstract = {Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.}, }
@article {pmid39702333, year = {2024}, author = {Liang, QZ and Chen, W and Liu, RC and Fu, QL and Fu, GH and Cheng, LF and Chen, HM and Jiang, NS and Zhu, T and Huang, Y}, title = {CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.}, journal = {Virology journal}, volume = {21}, number = {1}, pages = {322}, pmid = {39702333}, issn = {1743-422X}, support = {ZYTS202423//the Freedom Explore Program of Fujian Academy of Agricultural Sciences/ ; 2023J01363//the Natural Science Foundation Project of Fujian Province/ ; CARS-42//the National Waterfowl Industry Technology System of Modern Agriculture for China/ ; }, mesh = {*Circovirus/genetics/isolation & purification ; Animals ; *Ducks/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Sensitivity and Specificity ; Poultry Diseases/virology/diagnosis ; Recombinases/metabolism/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.
METHODS: A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 ℃ and allowed for visual interpretation without requiring sophisticated equipment.
RESULTS: The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45 min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.
CONCLUSION: These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.}, }
@article {pmid39701024, year = {2024}, author = {Lee, J and Jeong, C}, title = {Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.}, journal = {BMB reports}, volume = {}, number = {}, pages = {}, pmid = {39701024}, issn = {1976-670X}, abstract = {CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications.}, }
@article {pmid39700796, year = {2024}, author = {Wang, X and Ding, H and Sun, Y and Ma, Y and Wang, G and Chen, J and Choo, J and Chen, L}, title = {CRISPR/HCR-powered ratiometric fluorescence aptasensor for ochratoxin A detection.}, journal = {Food chemistry}, volume = {468}, number = {}, pages = {142437}, doi = {10.1016/j.foodchem.2024.142437}, pmid = {39700796}, issn = {1873-7072}, abstract = {To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH2) at 426 nm. The F426/F562 ratio serves as a "signal-on" indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %-3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %-110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.}, }
@article {pmid39700179, year = {2024}, author = {Bowland, K and Lai, J and Skaist, A and Zhang, Y and Teh, SSK and Roberts, NJ and Thompson, E and Wheelan, SJ and Hruban, RH and Karchin, R and Bailey, MH and Iacobuzio-Donahue, CA and Eshleman, JR}, title = {Islands of genomic stability in the face of genetically unstable metastatic cancer.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0298490}, pmid = {39700179}, issn = {1932-6203}, support = {P30 CA006973/CA/NCI NIH HHS/United States ; P50 CA062924/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Genomic Instability ; *Pancreatic Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Neoplasm Metastasis/genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/secondary ; Mutation ; Male ; Whole Genome Sequencing ; Female ; }, abstract = {INTRODUCTION: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9.
METHODS: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing.
RESULTS: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases.
CONCLUSIONS: Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.}, }
@article {pmid39700052, year = {2024}, author = {Li, X and Chen, B and Xie, Y and Luo, Y and Zhu, D and Wang, L and Su, S}, title = {Polyvalent Aptamers Structure-Mediated Fluorescent Aptasensor for the Early Diagnosis of Alzheimer's Disease by Coupling with HCR and CRISPR-Cas System.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c05329}, pmid = {39700052}, issn = {1520-6882}, abstract = {The early diagnosis of Alzheimer's disease (AD) plays a vital role in slowing the progression of AD and improving the quality of human life. However, it is still a challenge in the medical field. Herein, an ultrasensitive fluorescent aptasensor was designed for the detection of special phosphorylated tau181 (P-tau181) by coupling with polyvalent aptamers (PAs) structure, hybridization chain reaction (HCR), and the CRISPR-Cas system. Coupling with the signal amplification strategy, the specific recognition ability of the aptamer, and the high cleavage activity of Cas12a protein, the designed aptasensor showed a wide linear range (0.1-10[6] pg/mL), a low detection limit (0.069 pg/mL), high selectivity, and excellent anti-interference ability for the detection of P-tau181. Moreover, the aptasensor can efficiently analyze P-tau181 in artificial cerebro spinal fluid (aCSF) and serum, proving that it has a promising application in the early diagnosis of AD.}, }
@article {pmid39699588, year = {2025}, author = {Yang, Q and Dong, MJ and Xu, J and Xing, Y and Wang, Y and Yang, J and Meng, X and Xie, T and Li, Y and Dong, H}, title = {CRISPR/RNA Aptamer System Activated by an AND Logic Gate for Biomarker-Driven Theranostics.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {1}, pages = {169-180}, doi = {10.1021/jacs.4c08719}, pmid = {39699588}, issn = {1520-5126}, mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/analysis ; *Theranostic Nanomedicine ; Photosensitizing Agents/chemistry/pharmacology ; Photochemotherapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Biomarkers, Tumor ; Nanoparticles/chemistry ; }, abstract = {The development of an engineered RNA device capable of detecting multiple biomarkers to evaluate pathological states and autonomously implement responsive therapies is urgently needed. Here, we report InCasApt, an integrated nano CRISPR Cas13a/RNA aptamer theranostic platform capable of achieving both biomarker detection and biomarker-driven therapy. Within this system, a Cas13a/crRNA complex, a hairpin reporter (HR), a dinitroaniline caged Ce6 photosensitizer (Ce6-DN), and a DN-binding RNA aptamer precursor (DNBApt) are coloaded onto dendritic mesoporous silicon nanoparticles (DMSN) in a controlled manner. While InCasApt remains inert in normal cells, its programmable theranostic capabilities are activated in tumor cells that have elevated expression of carcinogenic miRNA-155 and miRNA-21. These miRNAs act as an AND logic gate, generating fluorescence for disease condition evaluation and ROS for photodynamic therapy. This process also upregulates antioncogene BRG1 and suppresses tumor migration by inhibiting the function of miRNA-155 and miRNA-21. These effects underscore the versatility of InCasApt as an miRNA-targeting strategy for bridging the gap between diagnosis and therapy.}, }
@article {pmid39699265, year = {2024}, author = {Li, X and Wang, L and Lin, J and Gu, Y and Liu, Z and Hu, J}, title = {Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0000924}, doi = {10.1128/spectrum.00009-24}, pmid = {39699265}, issn = {2165-0497}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (quinolones, macrolides, tetracyclines and carbapenems), and some of them were higher instead (aminoglycosides, clindamycins, rifampicin-associated, sulfonamides, methotrexates, beta-lactamases and ultrabroad-spectrum beta-lactamases). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.}, }
@article {pmid39699033, year = {2024}, author = {Paggi, RA and Ferrari, MC and Cerletti, M and Giménez, MI and Schwarz, TS and Marchfelder, A and De Castro, RE}, title = {Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.}, journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology}, volume = {}, number = {}, pages = {}, doi = {10.1002/bmb.21872}, pmid = {39699033}, issn = {1539-3429}, support = {//Universidad Nacional de Mar del Plata/ ; PICT 02228//Fondo para la Investigación Científica y Tecnológica (FONCYT)/ ; }, abstract = {Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).}, }
@article {pmid39698811, year = {2024}, author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA}, title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1192}, pmid = {39698811}, issn = {1362-4962}, support = {U19AI171110/NH/NIH HHS/United States ; U19NS132303/NS/NINDS NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; 2334028//National Science Foundation/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Panattoni Family Foundation/ ; //National Science Foundation Graduate Research Fellowship/ ; //Helen Hay Whitney Foundation/ ; //HHMI Fellow of The Jane Coffin Childs Fund for Medical Research/ ; DE-AC02-05CH11231//Department of Energy/ ; 24 180//Apple Tree Partners/ ; //Lawrence Livermore National Laboratory/ ; //UCB-Hampton University Summer Program/ ; //Mr. Li Ka Shing/ ; //Koret-Berkeley-TAU/ ; //Innovative Genomics Institute/ ; }, abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.}, }
@article {pmid39698318, year = {2024}, author = {Cao, S and Ma, D and Xie, J and Wu, Z and Yan, H and Ji, S and Zhou, M and Zhu, S}, title = {Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1474825}, pmid = {39698318}, issn = {2235-2988}, mesh = {*African Swine Fever Virus/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *African Swine Fever/diagnosis/virology ; *Point-of-Care Testing ; *Sensitivity and Specificity ; *Recombinases/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; }, abstract = {African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.}, }
@article {pmid39696686, year = {2024}, author = {Izadifar, M and Massumi, M and Prentice, KJ and Oussenko, T and Li, B and Elbaz, J and Puri, M and Wheeler, MB and Nagy, A}, title = {Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch.}, journal = {Stem cell research & therapy}, volume = {15}, number = {1}, pages = {486}, pmid = {39696686}, issn = {1757-6512}, support = {143231//Canadian Institutes of Health Research Foundation Grant/ ; 950-230422//Canada Research Chairs/ ; }, mesh = {*Insulin-Secreting Cells/metabolism/drug effects ; Animals ; Mice ; *Glucose/metabolism ; *Thymidine Kinase/genetics/metabolism ; Ganciclovir/pharmacology ; CRISPR-Cas Systems ; CDC2 Protein Kinase/metabolism/genetics ; Lab-On-A-Chip Devices ; Insulin/metabolism ; Homeodomain Proteins/metabolism/genetics ; Trans-Activators ; }, abstract = {BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch.
METHOD: We generated β cell-induced progenitor-like cells (βiPLCs) from primary mouse pancreatic β cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe βiPLC line, designated as FSβiPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FSβiPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip.
RESULTS: The βiPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The βiPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FSβiPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FSβiPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FSβiPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts.
CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells.}, }
@article {pmid39696608, year = {2024}, author = {Lin, Z and Yao, Q and Lai, K and Jiao, K and Zeng, X and Lei, G and Zhang, T and Dai, H}, title = {Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {311}, pmid = {39696608}, issn = {1474-760X}, mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics ; Gene Drive Technology/methods ; Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Humans ; }, abstract = {BACKGROUND: Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.
RESULTS: Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.
CONCLUSIONS: Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.}, }
@article {pmid39696488, year = {2024}, author = {Koonin, EV and Makarova, KS}, title = {CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {295}, pmid = {39696488}, issn = {1741-7007}, mesh = {*DNA Transposable Elements/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteria/genetics/virology ; Plasmids/genetics ; }, abstract = {CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition. Many plasmids carry type IV or subtype V-M CRISPR systems that appear to be involved in inter-plasmid competition. Numerous Tn7-like and Mu-like transposons encode CRISPR-associated transposases (CASTs) in which interference-defective CRISPR systems of type I or type V mediate RNA-guided, site-specific transposition. The recruitment of CRISPR systems and their components by MGE is a manifestation of extensive gene shuttling between host immune systems and MGE, a major trend in the coevolution of MGE with their hosts.}, }
@article {pmid39696352, year = {2024}, author = {Zheng, S and Luo, M and Huang, H and Huang, X and Peng, Z and Zheng, S and Tan, J}, title = {New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.}, journal = {Cell communication and signaling : CCS}, volume = {22}, number = {1}, pages = {592}, pmid = {39696352}, issn = {1478-811X}, support = {82460548//National Natural Science Foundation of China/ ; 20242BAB20368//Natural Science Foundation of Jiangxi Province of China/ ; }, mesh = {*Osteosarcoma/genetics/pathology ; *Mitophagy/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Bone Neoplasms/genetics/pathology/metabolism ; Neoplasm Metastasis/genetics ; Cell Line, Tumor ; Mice ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis ; Mitochondrial Precursor Protein Import Complex Proteins ; }, abstract = {BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.
METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.
RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.
CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making (https://mip-score.shinyapps.io/labtan/). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.}, }
@article {pmid39695426, year = {2024}, author = {Prado, MB and Coelho, BP and Iglesia, RP and Alves, RN and Boccacino, JM and Fernandes, CFL and Melo-Escobar, MI and Ayyadhury, S and Cruz, MC and Santos, TG and Beraldo, FH and Fan, J and Ferreira, FM and Nakaya, HI and Prado, MAM and Prado, VF and Duennwald, ML and Lopes, MH}, title = {Prion protein regulates invasiveness in glioblastoma stem cells.}, journal = {BMC cancer}, volume = {24}, number = {1}, pages = {1539}, pmid = {39695426}, issn = {1471-2407}, support = {2017/26158‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14952‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/03714-8//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/08198-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2020/07450‑5//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/14741‑9//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2019/11097‑1//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2017/20271‑0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 101796/2020‑0//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 409941/2021‑2//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 03592-2021 RGPIN//Natural Sciences and Engineering Research Council of Canada/ ; }, mesh = {*Glioblastoma/pathology/metabolism/genetics ; Humans ; *Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Neoplasm Invasiveness ; *Brain Neoplasms/pathology/metabolism/genetics ; Cell Proliferation ; Gene Knockout Techniques ; PrPC Proteins/metabolism/genetics ; Prion Proteins/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP[C]), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP[C] expression on GBM.
METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrP[C] knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP[C]-wild-type (WT) cells and PrP[C]-knockout (KO) cells were used to identify pathways regulated by PrP[C]. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP[C] in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.
RESULTS: Transcriptome analysis of PrP[C]-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP[C]-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP[C] disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP[C] levels through CD44 overexpression further emphasizes their regulatory role in these processes.
CONCLUSIONS: These findings establish PrP[C] as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.}, }
@article {pmid39694744, year = {2024}, author = {Zhu, Y and Yu, X and Wu, J}, title = {CRISPR/Cas: a toolkit for plant disease diagnostics.}, journal = {Trends in plant science}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tplants.2024.11.011}, pmid = {39694744}, issn = {1878-4372}, abstract = {Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.}, }
@article {pmid39694477, year = {2024}, author = {Harding, KR and Malone, LM and Kyte, NAP and Jackson, SA and Smith, LM and Fineran, PC}, title = {Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1194}, pmid = {39694477}, issn = {1362-4962}, support = {//Marsden Fund/ ; //Royal Society of New Zealand/ ; //Bioprotection Aotearoa/ ; //James Cook Research Fellowship/ ; //University of Otago Doctoral Scholarships/ ; //Division of Health Sciences Career Development Postdoctoral Fellowship/ ; //EMBO Postdoctoral Fellowship/ ; }, abstract = {The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.}, }
@article {pmid39693940, year = {2025}, author = {Baranova, SV and Zhdanova, PV and Golyshev, VM and Lomzov, AA and Pestryakov, PE and Chernonosov, AA and Koval, VV}, title = {Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex.}, journal = {Biochemical and biophysical research communications}, volume = {743}, number = {}, pages = {151176}, doi = {10.1016/j.bbrc.2024.151176}, pmid = {39693940}, issn = {1090-2104}, mesh = {*Thermodynamics ; *Molecular Dynamics Simulation ; *DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; *DNA, Single-Stranded/chemistry/metabolism ; *RNA/chemistry/metabolism ; Protein Binding ; Calorimetry ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry/metabolism ; Bacterial Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a's specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of -243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.}, }
@article {pmid39693439, year = {2024}, author = {Wang, Q and Xu, X and Chen, S and Lu, R and Li, L and Lo, CH and Liu, Z and Ning, K and Li, T and Kowal, TJ and Wang, B and Hartnett, ME and Wang, S and Qi, LS and Sun, Y}, title = {dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadn7540}, pmid = {39693439}, issn = {2375-2548}, mesh = {Animals ; *Retinitis Pigmentosa/therapy/genetics/pathology/metabolism ; *Disease Models, Animal ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; *Genetic Therapy/methods ; *Dependovirus/genetics ; Electroretinography ; Retinal Degeneration/therapy/genetics/pathology ; CRISPR-Cas Systems ; Humans ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Eye Proteins/metabolism/genetics ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; }, abstract = {Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6a[nmf363] mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.}, }
@article {pmid39693429, year = {2024}, author = {Shembrey, C and Yang, R and Casan, J and Hu, W and Chen, H and Singh, GJ and Sadras, T and Prasad, K and Shortt, J and Johnstone, RW and Trapani, JA and Ekert, PG and Fareh, M}, title = {Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadl0731}, pmid = {39693429}, issn = {2375-2548}, mesh = {*CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Gene Silencing ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Base Pair Mismatch ; Membrane Proteins/genetics ; Oncogenes ; RNA, Neoplasm/genetics/metabolism ; GTP Phosphohydrolases ; Proto-Oncogene Proteins B-raf ; }, abstract = {Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.}, }
@article {pmid39693343, year = {2024}, author = {Casagrande Raffi, G and Chen, J and Feng, X and Chen, Z and Lieftink, C and Deng, S and Mo, J and Zeng, C and Steur, M and Wang, J and Bleijerveld, OB and Hoekman, L and van der Wel, N and Wang, F and Beijersbergen, R and Zheng, J and Bernards, R and Wang, L}, title = {An antibiotic that mediates immune destruction of senescent cancer cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2417724121}, pmid = {39693343}, issn = {1091-6490}, support = {787925//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; LSH-TKI-LSHM20083//Health Holland/ ; 19-051-ASP//Mark Foundation For Cancer Research (The Mark Foundation for Cancer Research)/ ; 12539//Dutch Cancer Society KWF/ ; W2432050; 82372695//National Natural Science Foundation of China-Guangdong Joint Fund (NSFC)/ ; 2024A04J6484//Guangdong Basic and Applied Basic Research Foundation/ ; }, mesh = {Humans ; *Cellular Senescence/drug effects/immunology ; *Pyrans/pharmacology ; *Killer Cells, Natural/immunology/drug effects/metabolism ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Necroptosis/drug effects ; Neoplasms/immunology/drug therapy/metabolism ; CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism ; Anti-Bacterial Agents/pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics ; Pyroptosis/drug effects ; Apoptosis/drug effects ; Animals ; CRISPR-Cas Systems ; Polyether Polyketides ; }, abstract = {Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).}, }
@article {pmid39693337, year = {2024}, author = {Li, LL and Xiao, Y and Wang, B and Zhuang, Y and Chen, Y and Lu, J and Lou, Y and Li, R}, title = {A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2413564121}, pmid = {39693337}, issn = {1091-6490}, support = {32372551//MOST | National Natural Science Foundation of China (NSFC)/ ; 32402385//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFD1401100//MOST | National Key Research and Development Program of China (NKPs)/ ; NA//Max Planck Partner Group Program/ ; 226-2024-00159//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; }, mesh = {*Oryza/genetics/growth & development/parasitology/metabolism ; *Frameshift Mutation ; *Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; *Oxylipins/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Animals ; Plant Diseases/parasitology/genetics ; Hemiptera/genetics/growth & development/metabolism ; Gene Editing ; Signal Transduction ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; Gibberellins/metabolism ; }, abstract = {CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.}, }
@article {pmid39693330, year = {2024}, author = {Perampalam, P and McDonald, JI and Dick, FA}, title = {GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0315923}, pmid = {39693330}, issn = {1932-6203}, mesh = {Humans ; *Genes, Essential ; *CRISPR-Cas Systems ; *Workflow ; Ovarian Neoplasms/genetics/pathology ; Female ; Software ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.}, }
@article {pmid39692063, year = {2024}, author = {Yuan, P and Usman, M and Liu, W and Adhikari, A and Zhang, C and Njiti, V and Xia, Y}, title = {Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.}, journal = {Biotechnology journal}, volume = {19}, number = {12}, pages = {e202400457}, pmid = {39692063}, issn = {1860-7314}, support = {OHO01511//USDA Hatch Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Transformation, Genetic ; Biotechnology/methods ; Plants/genetics ; }, abstract = {Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.}, }
@article {pmid39690340, year = {2024}, author = {Dixit, Y and Yadav, P and Asnani, H and Sharma, AK}, title = {CRISPR/Cas9-Engineering for Increased Amylolytic Potential of Microbes for Sustainable Wastewater Treatment: A Review.}, journal = {Current microbiology}, volume = {82}, number = {1}, pages = {44}, pmid = {39690340}, issn = {1432-0991}, mesh = {*Amylases/biosynthesis/genetics ; Bacteria/enzymology/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; *Wastewater/chemistry/microbiology ; Water Purification/methods ; }, abstract = {Amylases are pivotal enzymes with extensive industrial applications, including food processing, textile manufacturing, pharmaceuticals, and biofuel production. Traditional methods for enhancing amylase production in microbial strains often lack precision and efficiency. The advent of CRISPR/Cas9 technology has revolutionized genetic engineering, offering precise and targeted modifications to microbial genomes. This review explores the potential of CRISPR/Cas9 for improving amylase production, highlighting its advantages over conventional methods. This review discusses the mechanism of CRISPR/Cas9, the identification and targeting of key genes involved in amylase synthesis and regulation, and the optimization of expression systems. Additionally, current review examines case studies demonstrating successful CRISPR/Cas9 applications in various microbial hosts. The review also delves into the integration of CRISPR/Cas9 in wastewater treatment, where genetically engineered amylolytic strains enhance the degradation of complex organic pollutants. Despite the promising prospects, challenges such as off-target effects and regulatory considerations remain. This review provides a comprehensive overview of the current advancements, challenges, and future directions in the application of CRISPR/Cas9 technology for amylase production and environmental biotechnology.}, }
@article {pmid39690326, year = {2024}, author = {Barber, HM and Pater, AA and Gagnon, KT and Damha, MJ and O'Reilly, D}, title = {Chemical engineering of CRISPR-Cas systems for therapeutic application.}, journal = {Nature reviews. Drug discovery}, volume = {}, number = {}, pages = {}, pmid = {39690326}, issn = {1474-1784}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.}, }
@article {pmid39688838, year = {2024}, author = {Xu, X and Zhang, Y and Liu, J and Wei, S and Li, N and Yao, X and Wang, M and Su, X and Jing, G and Xu, J and Liu, Y and Lu, Y and Cheng, J and Xu, Y}, title = {Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.4c13557}, pmid = {39688838}, issn = {1936-086X}, abstract = {The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.}, }
@article {pmid39687096, year = {2024}, author = {Karpenko, A and Shelenkov, A and Petrova, L and Gusarov, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V}, title = {Two multidrug-resistant Proteus mirabilis clones carrying extended spectrum beta-lactamases revealed in a single hospital department by whole genome sequencing.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40821}, pmid = {39687096}, issn = {2405-8440}, abstract = {Proteus mirabilis bacteria is a component of normal intestinal microflora of humans and animals, but can also be found in hospital settings causing urinary tract infections and sepsis. The problem of treating such infections is complicated by multidrug-resistant isolates producing extended spectrum beta-lactamases (ESBL), and the number of ESBL-carrying P. mirabilis strains has significantly increased recently. This study presents a detailed analysis of 12 multidrug-resistant P. mirabilis isolates obtained from the wounds of different patients in one surgical department of a multidisciplinary hospital in Moscow, Russia, using the short- and long-read whole genome sequencing. The isolates under investigation divided into two clusters (clones) C1 and C2 based on their genomic profiles and carried antimicrobial resistance (AMR) genes corresponding well with phenotypic profiles, which was the first case of reporting two different P. mirabilis clones obtained simultaneously from the same specimens at one hospital, to the best of our knowledge. Some genes, including ESBL encoding ones, were specific for either C1 or C2 (aac(6')-Ib10, ant(2″)-Ia, qnrA1, bla VEB-6 and fosA3, bla CTX -M-65 , correspondingly). Additionally, the Salmonella genomic islands 1 were found that differed in composition of multiple antibiotic resistance regions between C1 and C2 groups. CRISPR-Cas system type I-E was revealed only in C2 isolates, while the same set of virulence factors was determined for both P. mirabilis clones. Diversity of all genetic factors found in case of simultaneous existence of two clones collected from the same source at one department indicates high pathogenic potential of P. mirabilis and poses a requirement of proper spreading monitoring. The data obtained will facilitate the understanding of AMR transfer and dynamics within clinical P. mirabilis isolates and contribute to epidemiological surveillance of this pathogen.}, }
@article {pmid39686519, year = {2024}, author = {Hernandez, FJ}, title = {Nucleases: From Primitive Immune Defenders to Modern Biotechnology Tools.}, journal = {Immunology}, volume = {}, number = {}, pages = {}, doi = {10.1111/imm.13884}, pmid = {39686519}, issn = {1365-2567}, support = {2021-05641//Vetenskapsrådet/ ; //HORIZON-MSCA-2022-COFUND-101126600-SmartBRAIN3/ ; }, abstract = {The story of nucleases begins on the ancient battlefields of early Earth, where simple bacteria fought to survive against viral invaders. Nucleases are enzymes that degrade nucleic acids, with restriction endonucleases emerging as some of the earliest defenders, cutting foreign DNA to protect their bacteria hosts. However, bacteria sought more than just defence. They evolved the CRISPR-Cas system, an adaptive immune mechanism capable of remembering past invaders. The now-famous Cas9 nuclease, a key player in this system, has been harnessed for genome editing, revolutionising biotechnology. Over time, nucleases evolved from basic viral defence tools into complex regulators of immune function in higher organisms. In humans, DNases and RNases maintain immune balance by clearing cellular debris, preventing autoimmunity, and defending against pathogens. These enzymes have transformed from simple bacterial defenders to critical players in both human immunity and biotechnology. This review explores the evolutionary history of nucleases and their vital roles as protectors in the story of life's defence mechanisms.}, }
@article {pmid39684611, year = {2024}, author = {Pavlova, SV and Shulgina, AE and Minina, JM and Zakian, SM and Dementyeva, EV}, title = {Generation of Isogenic iPSC Lines for Studying the Effect of the p.N515del (c.1543_1545delAAC) Variant on MYBPC3 Function and Hypertrophic Cardiomyopathy Pathogenesis.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684611}, issn = {1422-0067}, support = {22-15-00271//Russian Science Foundation/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Humans ; *Myocytes, Cardiac/metabolism/cytology/pathology ; *Carrier Proteins/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; }, abstract = {The clinical significance of numerous cardiovascular gene variants remains to be determined. CRISPR/Cas9 allows for the introduction and/or correction of a certain variant in induced pluripotent stem cells (iPSCs). The resulting isogenic iPSC lines can be differentiated into cardiomyocytes and used as a platform to assess the pathogenicity of the variant. In this study, isogenic iPSC lines were generated for a variant of unknown significance found previously in a patient with hypertrophic cardiomyopathy (HCM), p.N515del (c.1543_1545delAAC) in MYBPC3. The deletion was corrected with CRISPR/Cas9 in the patient-specific iPSCs. The iPSC lines with the corrected deletion in MYBPC3 maintained pluripotency and a normal karyotype and showed no off-target CRISPR/Cas9 activity. The isogenic iPSC lines, together with isogenic iPSC lines generated earlier via introducing the p.N515del (c.1543_1545delAAC) variant in MYBPC3 of iPSCs of a healthy donor, were differentiated into cardiomyocytes. The cardiomyocytes derived from both panels of the isogenic iPSCs showed an increased size in the presence of the deletion in MYBPC3, which is one of the HCM traits at the cellular level. This finding indicates the effectiveness of these iPSC lines for studying the impact of the variant on HCM development.}, }
@article {pmid39684477, year = {2024}, author = {Borovikova, SE and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684477}, issn = {1422-0067}, support = {22-15-00381//Russian Science Foundation/ ; 075-15-2019-1661//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *RNA Polymerase II/metabolism/genetics ; Jurkat Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.}, }
@article {pmid39684395, year = {2024}, author = {Xuan, Q and Wang, J and Nie, Y and Fang, C and Liang, W}, title = {Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684395}, issn = {1422-0067}, support = {32201248//National Natural Science Foundation of China/ ; 242102111164//Key R&D and Promotion Projects in Henan Province/ ; 222301420106//Natural Science Foundation of Henan/ ; 222300420202//Natural Science Foundation of Henan/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Plants/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; }, abstract = {CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.}, }
@article {pmid39684387, year = {2024}, author = {Aksoy, MO and Bilinska, A and Stachowiak, M and Flisikowska, T and Szczerbal, I}, title = {Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684387}, issn = {1422-0067}, support = {2018/29/B/NZ2/00956//National Science Center/ ; }, mesh = {Animals ; *Adipogenesis/genetics ; *Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; *CRISPR-Cas Systems ; Swine ; *Gene Editing/methods ; Adipocytes/metabolism/cytology ; Gene Expression Regulation ; Lipid Metabolism/genetics ; }, abstract = {Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.}, }
@article {pmid39684256, year = {2024}, author = {Yang, P and Zhang, S and Hu, D and Li, X and Guo, Y and Guo, H and Zhang, L and Ding, X}, title = {Research Progress on the Mechanism and Application of the Type I CRISPR-Cas System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684256}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Archaea/genetics ; Bacteria/genetics ; Genetic Engineering/methods ; }, abstract = {The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.}, }
@article {pmid39684244, year = {2024}, author = {Wattad, H and Molcho, J and Manor, R and Weil, S and Aflalo, ED and Chalifa-Caspi, V and Sagi, A}, title = {Roadmap and Considerations for Genome Editing in a Non-Model Organism: Genetic Variations and Off-Target Profiling.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684244}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Palaemonidae/genetics ; Genetic Variation ; Polymorphism, Single Nucleotide ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Sex Chromosomes/genetics ; }, abstract = {The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved. Here, we demonstrated a generalized roadmap for a de novo genome annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed the typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For the genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome editing approach to embryos and the embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict the gene models by leveraging the available genomic, transcriptomic, and proteomic data, and enabling accurate gene prediction and guide design for knock-outs. A next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty.}, }
@article {pmid39683106, year = {2024}, author = {Zhan, X and Zhang, F and Li, N and Xu, K and Wang, X and Gao, S and Yin, Y and Yuan, W and Chen, W and Ren, Z and Yao, M and Wang, F}, title = {CRISPR/Cas: An Emerging Toolbox for Engineering Virus Resistance in Plants.}, journal = {Plants (Basel, Switzerland)}, volume = {13}, number = {23}, pages = {}, pmid = {39683106}, issn = {2223-7747}, support = {2023BBB044//Fei Wang/ ; 2022BBA0060//Fei Wang/ ; HBZY2023B004-3//Minghua Yao/ ; 2023HBSTX4-06//Ning Li/ ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future.}, }
@article {pmid39682777, year = {2024}, author = {Ryu, M and Yurube, T and Takeoka, Y and Kanda, Y and Tsujimoto, T and Miyazaki, K and Ohnishi, H and Matsuo, T and Kumagai, N and Kuroshima, K and Hiranaka, Y and Kuroda, R and Kakutani, K}, title = {Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.}, journal = {Cells}, volume = {13}, number = {23}, pages = {}, pmid = {39682777}, issn = {2073-4409}, support = {JP21K09323//Japan Society for the Promotion of Science/ ; JP24K02565//Japan Society for the Promotion of Science/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *TOR Serine-Threonine Kinases/metabolism ; *Proto-Oncogene Proteins c-akt/metabolism ; *Signal Transduction ; *RNA Interference ; *Intervertebral Disc Degeneration/therapy/genetics/metabolism/pathology ; Gene Silencing ; Phosphatidylinositol 3-Kinases/metabolism ; Nucleus Pulposus/metabolism/pathology ; Intervertebral Disc/metabolism/pathology ; Autophagy/genetics ; Regulatory-Associated Protein of mTOR/metabolism/genetics ; RNA, Small Interfering/metabolism/genetics ; Male ; Female ; Middle Aged ; }, abstract = {The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.}, }
@article {pmid39681230, year = {2024}, author = {Nafari, NB and Zamani, M and Mosayyebi, B}, title = {Recent advances in lateral flow assays for MicroRNA detection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120096}, doi = {10.1016/j.cca.2024.120096}, pmid = {39681230}, issn = {1873-3492}, abstract = {Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.}, }
@article {pmid39680881, year = {2025}, author = {Zeng, J and Huang, X and Yang, Y and Wang, J and Shi, Y and Li, H and Hu, N and Yu, B and Mu, J}, title = {Near-Infrared Optogenetic Nanosystem for Spatiotemporal Control of CRISPR-Cas9 Gene Editing and Synergistic Photodynamic Therapy.}, journal = {ACS applied materials & interfaces}, volume = {17}, number = {1}, pages = {701-710}, doi = {10.1021/acsami.4c18656}, pmid = {39680881}, issn = {1944-8252}, mesh = {*Photochemotherapy ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; *Infrared Rays ; Nanoparticles/chemistry ; Optogenetics/methods ; Cell Line, Tumor ; Neoplasms/therapy/drug therapy/genetics ; Photosensitizing Agents/chemistry/pharmacology ; Mice, Inbred BALB C ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Mice, Nude ; }, abstract = {Controlling CRISPR/Cas9 gene editing at the spatiotemporal resolution level, especially for in vivo applications, remains a great challenge. Here, we developed a near-infrared (NIR) light-activated nanophotonic system (UCPP) for controlled CRISPR-Cas9 gene editing and synergistic photodynamic therapy (PDT). Lanthanide-doped upconversion nanoparticles are not only employed as carriers for intracellular plasmid delivery but also serve as the nanotransducers to convert NIR light (980 nm) into visible light with emission at 460 and 650 nm, which could result in simultaneous activation of gene editing and PDT processes, respectively. Such unique design not only achieves light-controlled precise gene editing of hypoxia-inducible factor 1α with minimal off-target effect, which effectively ameliorates the hypoxic state at tumor sites, but also facilitates the deep-seated PDT process with synergistic antitumor effect. This optogenetically activatable CRISPR-Cas9 nanosystem holds great potential for spatially controlled in vivo gene editing and targeted cancer therapy.}, }
@article {pmid39680738, year = {2024}, author = {Yang, S and Hu, G and Wang, J and Song, J}, title = {CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00632}, pmid = {39680738}, issn = {2161-5063}, abstract = {In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.}, }
@article {pmid39680576, year = {2024}, author = {Guguin, J and Chen, TY and Cuinat, S and Besson, A and Bertiaux, E and Boutaud, L and Ardito, N and Imaz Murguiondo, M and Cabet, S and Hamel, V and Thomas, S and Pain, B and Edery, P and Putoux, A and Tang, TK and Mazoyer, S and Delous, M}, title = {A Taybi-Linder syndrome-related RTTN variant impedes neural rosette formation in human cortical organoids.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011517}, pmid = {39680576}, issn = {1553-7404}, mesh = {Humans ; *Organoids/metabolism ; *Microcephaly/genetics ; *Neural Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Cell Cycle Proteins/genetics/metabolism ; RNA, Small Nuclear/genetics/metabolism ; Centrosome/metabolism ; CRISPR-Cas Systems ; Mutation ; Exome Sequencing ; Cerebral Cortex/metabolism ; }, abstract = {Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly.}, }
@article {pmid39680522, year = {2024}, author = {Zheng, J and Li, B and Jia, L and Zhang, J and Gong, Z and Le, Y and Nian, X and Li, X and Liu, B and Yu, D and Zhang, Z and Li, C}, title = {Tumorigenicity decrease in Bcl-xL deficient MDCK cells ensuring the safety for influenza vaccine production.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0311069}, pmid = {39680522}, issn = {1932-6203}, mesh = {Animals ; Dogs ; *bcl-X Protein/genetics/metabolism ; Madin Darby Canine Kidney Cells ; *Influenza Vaccines ; Mice ; *Apoptosis ; Cell Proliferation ; CRISPR-Cas Systems ; Mice, Nude ; Carcinogenesis/genetics ; Cell Movement ; Gene Editing ; Mice, Inbred BALB C ; }, abstract = {Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells' tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.}, }
@article {pmid39680038, year = {2025}, author = {Van, R and Pan, X and Rostami, S and Liu, J and Agarwal, PK and Brooks, B and Rajan, R and Shao, Y}, title = {Exploring CRISPR-Cas9 HNH-Domain-Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation.}, journal = {Biochemistry}, volume = {64}, number = {1}, pages = {289-299}, doi = {10.1021/acs.biochem.4c00651}, pmid = {39680038}, issn = {1520-4995}, mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; *Quantum Theory ; Molecular Dynamics Simulation ; Thermodynamics ; DNA/chemistry/metabolism/genetics ; Catalytic Domain ; CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; }, abstract = {The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and co-workers (Nierzwicki et al., Nat. Catal. 2022 5, 912) using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments. For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.}, }
@article {pmid39679901, year = {2025}, author = {Zhang, W and Li, Z and Lun, X and Guo, Y}, title = {Telomerase-Responsive CRISPR System-Regulated Nanobomb for Triggering Research on Telomerase "Self-Detonation".}, journal = {ACS applied materials & interfaces}, volume = {17}, number = {1}, pages = {725-738}, doi = {10.1021/acsami.4c18859}, pmid = {39679901}, issn = {1944-8252}, mesh = {*Telomerase/metabolism/genetics ; Humans ; Animals ; Mice ; MCF-7 Cells ; *Aptamers, Nucleotide/chemistry ; Female ; CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Mice, Nude ; Silicon Dioxide/chemistry ; Naphthalenes/chemistry/pharmacology ; Breast Neoplasms/pathology/metabolism ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/chemistry ; Cell Proliferation/drug effects ; Aminobenzoates ; }, abstract = {Targeting tumor markers is one of the most important approaches to tumor therapy, and the "suicide" pattern of tumor marker response is a very challenging study. Telomerase, as one of the key factors associated with human longevity and cancer progression, is considered to be an emerging biomarker for cancer diagnosis. The targeted drug delivery nanobomb─BIBR1532@HSN/FQDNA/MUC1 aptamer (B@HDA) is prepared in this study based on hollow silica nanoparticles (HSN) and CRISPR systems. Amino-modified FQDNA and amino-modified MUC1 aptamer are covalently attached to the surface of carboxyl-functionalized HSN. The modified MUC1 aptamer directs the nanobomb to specifically target breast cancer cells (MCF-7) and FQDNA sequesters the telomerase inhibitor (BIBR1532) within the HSN. Telomerase primers (TPs) is recognized by the highly expressed telomerase in MCF-7 cells and is elongated to form DNA substrates. The substrate pairs with crRNA bases to effectively activate CRISPR-Cas12a. The activated CRISPR-Cas12a precisely cut FQDNA, releasing BIBR1532, which inhibits telomerase activity. This strategy achieves telomerase "suicide". The nanobomb described above has the following advantages. (1) The "closing" effect of FQDNA contributes to reducing the nonspecific release of BIBR1532. (2) B@HDA, combined with CRISPR, regulates mitochondrial dysfunction and cell senescence in MCF-7 cells. (3) In the tumor-bearing mouse model, B@HDA, combined with CRISPR, exhibits good biocompatibility and an obvious tumor ablation effect on MCF-7 tumors, suggesting potential application prospects across a wide range of cancer cell lines. In summary, the proposed nanobomb provides a tunable switch approach for the specific inhibition of telomerase and the reduction of tumor cell growth, representing a promising avenue for promoting senescence and treating cancer.}, }
@article {pmid39678914, year = {2024}, author = {Rostampour, M and Panahi, B and Masoumi Jahandizi, R}, title = {The CRISPR-Cas system in Lactiplantibacillus plantarum strains: identification and characterization using a genome mining approach.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1394756}, pmid = {39678914}, issn = {1664-302X}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system against bacteriophages. In this study, 675 sequences of Lactiplantibacillus plantarum isolates deposited in GenBank were analyzed in terms of diversity, occurrence, and evolution of the CRISPR-Cas system. This study investigated the presence, structural variations, phylogenetic relationships, and diversity of CRISPR-Cas systems in 675 L. plantarum strains. The analysis revealed that 143 strains harbor confirmed CRISPR-Cas systems, with subtype II-A being predominant. Moreover, targeting phages and plasmid diversity between the predicted systems were dissected. The results indicated that approximately 22% of the isolates with verified and complete CRISPR systems exhibited the coexistence of both subtypes II-A and I-E within their genomes. The results further showed that in subtype II-A, the length of the repeat sequence was 36 nucleotides, on average. In addition, the number of spacers in subtypes II-A and I-E varied between 1-24 and 3-16 spacers, respectively. The results also indicated that subtype II-A has nine protospacer adjacent motifs, which are 5'-CC-3', 5'-GAA-3', 5'-TGG-3', 5'-CTT-3', 5'-GGG-3', 5'-CAT-3', 5'-CTC-3', 5'-CCT-3', and 5'-CGG-3'. In addition, the identified systems displayed a potential for targeting Lactobacillus phages. The investigation of the relationship between the targeting of Lactobacillus phages by the antiphage system in L. plantarum species showed that subtype II-A had the highest diversity in targeting Lactobacillus phages than subtype I-E. In conclusion, current findings offer a perspective on the prevalence and evolution of the CRISPR-Cas system in L. plantarum, contributing novel insights to the expanding field of CRISPR-Cas systems within lactobacillus strains. This knowledge establishes a foundation for future applied studies focused on enhancing phage resistance in industrial fermentation, reducing contamination risks, and improving product quality. The identified targeting diversity may also foster advancements in phage therapy through the development of CRISPR-based antimicrobials.}, }
@article {pmid39677770, year = {2024}, author = {Hoelting, K and Madlmayr, A and Hoeger, B and Lewitz, D and Weng, M and Haider, T and Duggan, M and Ross, R and Horgen, FD and Sperandio, M and Dietrich, A and Gudermann, T and Zierler, S}, title = {TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39677770}, issn = {2692-8205}, support = {P20 GM103466/GM/NIGMS NIH HHS/United States ; U54 GM138062/GM/NIGMS NIH HHS/United States ; }, abstract = {T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg[2+] homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg[2+] homeostasis, Ca[2+] signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (Treg) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg[2+] homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases.}, }
@article {pmid39677748, year = {2024}, author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Fischer, KD and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Wang, E and Imig, J}, title = {RNA Binding of GAPDH Controls Transcript Stability and Protein Translation in Acute Myeloid Leukemia.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.12.02.626357}, pmid = {39677748}, issn = {2692-8205}, abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.}, }
@article {pmid39676682, year = {2024}, author = {Chen, J and Lin, X and Xiang, W and Chen, Y and Zhao, Y and Huang, L and Liu, L}, title = {DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1241}, pmid = {39676682}, issn = {1362-4962}, support = {32022047//National Natural Science Foundation of China/ ; 3502Z20227020//Natural Science Foundation of Xiamen, China/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; }, abstract = {Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.}, }
@article {pmid39676076, year = {2024}, author = {Gao, Z and Bak, RO}, title = {Integration of large genetic payloads using prime editing and site-specific integrases.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, pmid = {39676076}, issn = {1750-2799}, support = {101041231//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; R238-2016-3349//Lundbeckfonden (Lundbeck Foundation)/ ; 8056-00010B//Innovationsfonden (Innovation Fund Denmark)/ ; NNF21OC0071259//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; }, }
@article {pmid39675005, year = {2024}, author = {Rubio, A and Garzón, A and Moreno-Rodríguez, A and Pérez-Pulido, AJ}, title = {Biological warfare between two bacterial viruses in a defense archipelago sheds light on the spread of CRISPR-Cas systems.}, journal = {Cell reports}, volume = {43}, number = {12}, pages = {115085}, doi = {10.1016/j.celrep.2024.115085}, pmid = {39675005}, issn = {2211-1247}, mesh = {*CRISPR-Cas Systems/genetics ; Acinetobacter baumannii/genetics ; Biological Warfare ; Genome, Bacterial ; }, abstract = {CRISPR-Cas systems are adaptive immunity systems of bacteria and archaea that prevent infection by viruses and other external mobile genetic elements. It is currently known that these defense systems can be co-opted by the same viruses. We have found one of these viruses in the opportunistic pathogen Acinetobacter baumannii, and the same system has been also found in an integration hotspot of the bacterial genome that harbors other multiple defense systems. The CRISPR-Cas system appears to especially target another virus that could compete with the system itself for the same integration site. This virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this viral warfare involving antiviral systems could be useful in the fight against infections caused by bacteria, and it would also shed light on how CRISPR-Cas systems expand in bacteria.}, }
@article {pmid39673958, year = {2025}, author = {Yang, R and Guan, X and Zhang, J and Moon, J and Guo, C and Jia, Z and Hou, C and Ganbaatar, U and Tricarico, S and Ma, AWK and Liu, C}, title = {Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe.}, journal = {Biosensors & bioelectronics}, volume = {271}, number = {}, pages = {117054}, pmid = {39673958}, issn = {1873-4235}, support = {U01 CA269147/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; *Human papillomavirus 16/genetics/isolation & purification ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; DNA, Single-Stranded/chemistry ; DNA Probes/chemistry/genetics ; Carbocyanines/chemistry ; Lab-On-A-Chip Devices ; Papillomavirus Infections/diagnosis/virology ; CRISPR-Associated Proteins/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Rapid, sensitive, and specific nucleic acid detection methods play crucial roles in clinical diagnostics and healthcare. Here, we report a novel amphiphilic DNA fluorescence probe for CRISPR-based nucleic acid detection. Unlike conventional fluorophore-quencher probe detection system, our amphiphilic DNA fluorescence probe features a hydrophobic Cy5 fluorophore head and a hydrophilic single-stranded DNA (ssDNA) tail. By combining the amphiphilic DNA fluorescence probe with a paper-based microfluidic device, we developed a quencher-free, CRISPR-based detection system for target nucleic acid quantification. In the presence of the target nucleic acid, the activated CRISPR-Cas12a enzyme cleaves the hydrophilic ssDNA tail of the amphiphilic DNA fluorescence probe, releasing the hydrophobic Cy5 head and altering the wettability of the CRISPR reaction solution. When the CRISPR reaction product is applied to the paper-based microfluidic device, the migration of the cleaved Cy5 head along the hydrophilic microfluidic channel is limited. The higher the target nucleic acid concentration, the shorter the fluorescence diffusion distance, enabling visual quantification of the nucleic acid target. We used human papillomavirus-16 (HPV-16) DNA as a model to evaluate the analytical performance of the system. Furthermore, we validated its clinical feasibility by testing clinical swab samples, achieving results comparable to the traditional PCR method. Our quencher-free CRISPR-based detection system shows potential for simple, affordable, and sensitive clinical diagnostics of HPV-associated cancer and other infectious diseases.}, }
@article {pmid39656986, year = {2024}, author = {Jordan, B}, title = {[New-generation editing].}, journal = {Medecine sciences : M/S}, volume = {40}, number = {11}, pages = {869-871}, doi = {10.1051/medsci/2024149}, pmid = {39656986}, issn = {1958-5381}, mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems ; Animals ; Bacteria/genetics ; }, abstract = {Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.}, }
@article {pmid39673266, year = {2024}, author = {Molina-Sánchez, MD and Martínez-Abarca, F and Millán, V and Mestre, MR and Stehantsev, P and Stetsenko, A and Guskov, A and Toro, N}, title = {Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14229-14243}, pmid = {39673266}, issn = {1362-4962}, support = {P20_00047//Consejeria de Transformacion Economica, industria, Conocimiento y Universidades/ ; //ERDF A way of making Europe/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Adaptive Immunity/genetics ; Bacterial Proteins/genetics/metabolism ; Gastrointestinal Microbiome/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics/immunology ; RNA Interference ; }, abstract = {Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.}, }
@article {pmid39672387, year = {2025}, author = {He, X and Li, P and Cao, H and Zhang, X and Zhang, M and Yu, X and Sun, Y and Ghonaim, AH and Ma, H and Li, Y and Shi, K and Zhu, H and He, Q and Li, W}, title = {Construction of a recombinant African swine fever virus with firefly luciferase and eGFP reporter genes and its application in high-throughput antiviral drug screening.}, journal = {Antiviral research}, volume = {233}, number = {}, pages = {106058}, doi = {10.1016/j.antiviral.2024.106058}, pmid = {39672387}, issn = {1872-9096}, mesh = {Animals ; *African Swine Fever Virus/genetics/drug effects ; Chlorocebus aethiops ; *Green Fluorescent Proteins/genetics ; *Antiviral Agents/pharmacology ; Vero Cells ; *Genes, Reporter ; Swine ; *Luciferases, Firefly/genetics ; *African Swine Fever/virology ; *Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Virus Replication/drug effects ; CRISPR-Cas Systems ; Gene Editing/methods ; Macrophages, Alveolar/virology/drug effects ; }, abstract = {African Swine Fever (ASF) is a highly lethal and contagious disease in pigs caused by African Swine Fever Virus (ASFV), which primarily infects domestic pigs and wild boars, with a mortality rate of up to 100%. Currently, there are no commercially available vaccines or drugs that are both safe and effective against ASFV. The ASFV 0428C strain was continuously passaged in Vero cells, and the adapted ASFV demonstrated efficient replication in Vero cells. The adapted ASFV was used as the parental virus, and an expression cassette encoding a dual reporter gene for firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP) was inserted into the ASFV genome using CRISPR/Cas9 gene editing technology to construct a recombinant ASFV variant (rASFV-FLuc-eGFP). rASFV-Fluc-eGFP was genetically stable, effectively infected porcine alveolar macrophages (PAM) and Vero cells, and expressed Fluc and eGFP concurrently. This study provides a tool for investigating the infection and pathogenic mechanisms of ASFV, as well as for screening essential host genes and antiviral drugs. Additionally, a high-throughput screening model of antiviral drugs was established based on rASFV-FLuc-eGFP in passaged cells, 218 compounds from the FDA-approved compound library were screened, and 5 candidate compounds with significant inhibitory effects in Vero cells were identified. The inhibitory effects on ASFV were further validated in both Vero and PAM cells, resulting in the identification of Salvianolic acid C (SAC), which demonstrated inhibitory effects and safety in both cell types. SAC is a candidate drug for the prevention and control of ASFV and shows promising application prospects.}, }
@article {pmid39671433, year = {2024}, author = {Taylor, T and Zhu, HV and Moorthy, SD and Khader, N and Mitchell, JA}, title = {The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011513}, pmid = {39671433}, issn = {1553-7404}, support = {R01 HG010045/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Mice ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; *Left-Right Determination Factors/genetics/metabolism ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Developmental ; }, abstract = {Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.}, }
@article {pmid39671335, year = {2024}, author = {Kolodziejczyk, A and Hoffmann, J and Cubillos, P and Albert, M}, title = {Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/67598}, pmid = {39671335}, issn = {1940-087X}, mesh = {Humans ; *Electroporation/methods ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Cerebral Cortex/cytology ; Gene Knockout Techniques/methods ; Neocortex/cytology ; }, abstract = {Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.}, }
@article {pmid39671009, year = {2024}, author = {Yu, Y and Li, Q and Yu, H and Li, Q}, title = {Comparative Analysis of Promoter Activity in Crassostrea gigas Embryos: Implications for Bivalve Gene Editing.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {27}, number = {1}, pages = {20}, pmid = {39671009}, issn = {1436-2236}, support = {42276111//National Natural Science Foundation of China/ ; ZR2022MC171//Natural Science Foundation of Shandong Province/ ; }, mesh = {Animals ; *Crassostrea/genetics/embryology ; *Promoter Regions, Genetic ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; *Histones/metabolism/genetics ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Peptide Elongation Factor 1/genetics/metabolism ; Aquaculture ; }, abstract = {In recent years, CRISPR/Cas9 gene editing technology has emerged as a powerful genetic tool with potential application in aquaculture. Crassostrea gigas, as a valuable species in aquaculture, holds promising potential for genetic enhancement and breeding through gene editing. However, the lack of efficient promoters for driving exogenous gene expression poses a major obstacle in bivalve gene editing. In this study, we isolated the promoter sequences of the β-tub and histone H3.3A genes from C. gigas. DNA expression constructs were generated by linking the promoters with the enhanced green fluorescent protein (EGFP) reporter and compared with the promoter activity of the endogenous EF-1α gene and an exogenous OsHV-1 promoter in C. gigas embryos. All four promoters effectively drive the expression of EGFP during early embryonic development in C. gigas. Among these four promoters, the β-tub promoter is the most potent promoter in driving EGFP expression in C. gigas embryos as early as 4.5 h after fertilization. The OsHV-1 promoter showed similar activity as β-tub promoter and appeared to be more active than the EF-1α and histone H3.3A promoters in C. gigas embryos. Furthermore, we assessed their performance in other three C. gigas relatives (Crassostrea ariakensis, Crassostrea nippona, and Crassostrea sikamea) and similar results were found. Collectively, these data suggest that the β-tub promoter is an effective promoter in directing gene expression in directing gene expression in oyster embryos, thus offering a potential application for gene editing in bivalves.}, }
@article {pmid39670656, year = {2024}, author = {Jung, JK and Dreyer, KS and Dray, KE and Muldoon, JJ and George, J and Shirman, S and Cabezas, MD and d'Aquino, AE and Verosloff, MS and Seki, K and Rybnicky, GA and Alam, KK and Bagheri, N and Jewett, MC and Leonard, JN and Mangan, NM and Lucks, JB}, title = {Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00469}, pmid = {39670656}, issn = {2161-5063}, support = {R01 EB026510/EB/NIBIB NIH HHS/United States ; }, abstract = {Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.}, }
@article {pmid39670632, year = {2024}, author = {Chen, W and Liu, L and Cheng, L}, title = {Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00695}, pmid = {39670632}, issn = {2161-5063}, abstract = {CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.}, }
@article {pmid39670384, year = {2025}, author = {Blennemann, M and Verma, A and Bachl, S and Carnevale, J and Engelhardt, BE}, title = {Understanding TCR T cell knockout behavior using interpretable machine learning.}, journal = {Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing}, volume = {30}, number = {}, pages = {382-393}, pmid = {39670384}, issn = {2335-6936}, mesh = {Humans ; *Receptors, Antigen, T-Cell/immunology/genetics ; *Machine Learning ; *Computational Biology ; *T-Lymphocytes/immunology ; *Neoplasms/immunology/genetics ; Gene Knockout Techniques ; Neural Networks, Computer ; CRISPR-Cas Systems ; }, abstract = {Genetic perturbation of T cell receptor (TCR) T cells is a promising method to unlock better TCR T cell performance to create more powerful cancer immunotherapies, but understanding the changes to T cell behavior induced by genetic perturbations remains a challenge. Prior studies have evaluated the effect of different genetic modifications with cytokine production and metabolic activity assays. Live-cell imaging is an inexpensive and robust approach to capture TCR T cell responses to cancer. Most methods to quantify T cell responses in live-cell imaging data use simple approaches to count T cells and cancer cells across time, effectively quantifying how much space in the 2D well each cell type covers, leaving actionable information unexplored. In this study, we characterize changes in TCR T cell's interactions with cancer cells from live-cell imaging data using explainable artificial inte