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Found: 26 139
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The synergistic effect of DNA nanostructures and CRISPR/Cas system for cancer diagnosis and treatment.
Cancer remains one of the most formidable global public health challenges, exerting a profound and detrimental impact on human health. Despite substantial advancements in cancer research, the escalating incidence and mortality rates underscore the persistent and growing burden on global healthcare systems. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, heralded as a revolutionary gene-editing tool, holds immense promise for cancer treatment. However, its efficacy is critically contingent upon developing efficient delivery strategies. DNA nanocarriers, characterized by their programmability, sequence specificity, and design flexibility, emerge as a highly effective vehicle for delivering the CRISPR/Cas system, facilitating the precise transportation of gene-editing tools to the cell nucleus. The integration of DNA nanocarriers with CRISPR/Cas technology provides a new paradigm for precise and controllable gene editing. Through programmable spatial assembly, DNA nanocarriers can protect Cas9 ribonucleoprotein complexs (RNPs), facilitate endosomal escape, and co-localize donor DNA to promote homology-directed repair. These synergistic effects bridge molecular programmability and genetic functionality, paving the way for safer and more efficient genome engineering. This review aims to evaluate the application of DNA nanocarriers in cancer diagnosis comprehensively and to explore their potential utility in cancer therapy when combined with the CRISPR/Cas system, offering novel insights and significant scientific contributions to the field.
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Monoclonal antibodies production in microbial systems: Current status, challenges and perspectives.
Monoclonal antibodies (mAbs) serve as indispensable tools in diagnostics, clinical therapeutics, and biomedical research. However, their large-scale production faces significant challenges due to the high costs and lengthy timelines associated with conventional mammalian cell-based expression systems. Microbial expression platforms have emerged as a transformative alternative, offering cost-effectiveness, rapid cultivation cycles, and superior genetic tractability for industrial-scale monoclonal antibodies production. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing enable precise metabolic engineering of host strains to enhance protein folding, secretion efficiency, and translational accuracy. Synthetic biology approaches facilities the reconstruction of mammalian glycosylation pathways in microbial systems, yielding monoclonal antibodies with near-native structural integrity. Furthermore, AI (artificial intelligence)-driven optimization of expression vectors, promoter systems, and culture conditions, combined with high-throughput screening of engineered strains, significantly accelerates the identification of high-yield production clones. This review comprehensively examines current progress in microbial expression systems, strain engineering strategies, and fermentation optimization for enhanced monoclonal antibodies production, while critically discussing existing limitations and potential solutions to advance the field.
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A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors.
The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.
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Safe and Efficient CRISPR Genome Editing of Primary Human T Cells Using a Droplet-Based Cell Mechanoporation Platform.
T cell engineering is a transformative strategy for adoptive cell therapy, holding the key to treating a wide array of human diseases. However, clinical translation is limited by current intracellular delivery methods that compromise viability, induce stress responses, and restrict scalability. This study presents a microfluidic droplet mechanoporation system tailored for primary human T cells, enabling efficient, stable, and clinically scalable gene delivery. Delivery of 2000 kDa fluorescein isothiocyanate (FITC)-dextran achieves ∼98% efficiency and >90% post-treatment viability, even at high cell densities, supporting the rapid production of therapeutically relevant cell numbers. The platform efficiently delivers mRNA, achieving transfection efficiencies approaching 99%; further, chimeric antigen receptor (CAR)-encoding mRNA is successfully delivered to generate CAR-expressing T cells with tunable surface expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins are effectively delivered for both single and multiplex knockouts (TRAC and PDCD-1), achieving up to a 2.35-fold higher efficiency than electroporation. Longitudinal analyses confirm preserved viability, proliferation, genome integrity, and T cell phenotypic stability. Collectively, these results establish microfluidic droplet mechanoporation as a safe, efficient, and scalable platform for the clinical manufacturing of engineered T cell therapies.
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Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger.
Since the first CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system was developed for creating double-stranded DNA breaks, it has been adapted and improved for different biotechnological applications. In this issue of The FEBS Journal, Arentshorst et al. developed a novel approach to enhance transgene expression of a specific protein, patulin synthase (PatE) from Penicillium expansum, in the important industrial filamentous fungus Aspergillus niger. Their technique involved the disruption of selected genes with counter-effects on targeted protein production and simultaneous integration of glucoamylase landing sites into the disrupted gene locus such as protease regulator (prtT) in an ATP-dependent DNA helicase II subunit 1 (kusA or ku70)-deletion strain. Multiple copies of the PatE transgene expression cassette were introduced by CRISPR-Cas9-mediated insertion. The purified PatE was further used for structural and functional studies, and the technique laid the foundation for elevating the overall production of various proteins or chemicals in those industrially important fungi.
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Chemical and optical control of CRISPR-associated nucleases.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system of bacteria has furnished programmable nucleases (e.g., Cas9) that are transforming the field of genome editing with applications in basic and biomedical research, biotechnology, and agriculture. However, broader real-world applications of Cas9 require precision control of its activity over dose, time, and space as off-target effects, embryonic mosaicism, chromosomal translocations, and genotoxicity have been observed with elevated and/or prolonged nuclease activity. Here, we review chemical and optical methods for precision control of Cas9's activity.
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Recent advances in generation of doubled haploid plants for genetic improvement in solanaceous vegetable crops.
Genotype-specific protocols and advancements focusing on CRISPR/Cas9-based haploid induction for doubled haploid (DH) production are poised to revolutionize plant breeding for faster genetic improvement in solanaceous crops. The need for swift development of improved cultivars offering greater resilience to biotic and abiotic stresses stems from the emergence of climate change risks. While conventional methods are effective, novel methods for precise crop genome manipulation are required. The in vivo and in vitro protocols leading to fixation of homozygosity and rapid attainment of homozygous DH (doubled haploids) lines have led to a resurgence in research on haploids and DH. The efforts for haploid production have been primarily concentrated on in vitro androgenesis and gynogenesis. The success of these in vitro techniques depends on various parameters, including culture conditions, developmental stage of the microspore, pretreatment, culture medium, growth regulators, and other media additives. Breakthroughs in genome-editing technologies, such as the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, have provided a new avenue for exploring in vivo haploid embryogenesis for haploid induction by means of haploid inducer factors, such as DMP (DMP domain of unknown function 679 membrane protein), ECS (egg cell-specific), MTL (matrilineal), and BBM (Baby boom). A successful haploid induction system in vegetable crops will be possible through the development of efficient in vivo and in vitro androgenesis and gynogenesis protocols. For genome-editing to be feasible, protocols must be optimized for commercially significant solanaceous crops like pepper, potatoes, eggplant, and tomatoes. The existing protocols for genome doubling need to be further improved for solanaceous crops. These developments are pivotal for the advancement and harnessing of haploid technology to its full potential in crop breeding.
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CRISPR-CasRx knock-in mice for RNA degradation.
The RNA editing tool CRISPR-CasRx has provided a platform for a range of transcriptome analysis tools and therapeutic approaches with its broad efficacy and high specificity. To enable the application of CasRx in vivo, we established a Credependent CasRx knock-in mouse. Using these mice, we specifically knocked down the expression of Meis1 and Hoxb13 in cardiomyocytes, which induced cardiac regeneration after myocardial infarction. We also knocked down the lncRNA Mhrt in cardiomyocytes with the CasRx knock-in mice, causing hypertrophic cardiomyopathy. In summary, we generated a Credependent CasRx knock-in mouse that can efficiently knock down coding gene and lncRNA expression in specific somatic cells. This in vivo CRISPR-CasRx system is promising for gene function research and disease modeling.
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Effective regeneration of dystrophic muscle using autologous iPSC-derived progenitors with CRISPR-Cas9 mediated precise correction.
Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by a lack of dystrophin, which eventually leads to apoptosis of muscle cells and impaired muscle contractility. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) gene editing of induced pluripotent stem cells (IPSC) offers the potential to correct the DMD gene defect and create healthy IPSC for autologous cell transplantation without causing immune activation. However, IPSC carry a risk of tumor formation, which can potentially be mitigated by differentiation of IPSC into myogenic progenitor cells (MPC). We hypothesize that precise genetic editing in IPSC using CRISPR-Cas9 technology, coupled with MPC differentiation and autologous transplantation, can lead to safe and effective muscle repair. With future research, our hypothesis may provide an optimal autologous stem cell-based approach to treat the dystrophic pathology and improve the quality of life for patients with DMD.
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Nrf2-Dependent and -Independent Effects of tert-Butylhydroquinone, CDDO-Im, and H2O2 in Human Jurkat T Cells as Determined by CRISPR/Cas9 Gene Editing.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a stress-activated transcription factor activated by stimuli such as electrophilic compounds and other reactive xenobiotics. Previously, we have shown that the commonly used food additive and Nrf2 activator tert-butylhydroquinone (tBHQ) suppresses interleukin-2 (IL-2) production, CD25 expression, and NFκB activity in human Jurkat T cells. The purpose of the current studies was to determine whether these effects were dependent upon Nrf2 by developing a human Nrf2-null T cell model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technology. The current studies show that suppression of CD25 expression by tBHQ is partially dependent on Nrf2, whereas inhibition of IL-2 secretion is largely Nrf2-independent. Interestingly, tBHQ inhibited NFκB activation in an Nrf2-independent manner. This was an unexpected finding since Nrf2 inhibits NFκB activation in other models. These results led us to investigate another more potent Nrf2 activator, the synthetic triterpenoid 1[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im). Treatment of wild-type and Nrf2-null Jurkat T cells with CDDO-Im resulted in an Nrf2-dependent suppression of IL-2. Furthermore, susceptibility to reactive oxygen species was significantly enhanced in the Nrf2-null clones as determined by decreased mitochondrial membrane potential and cell viability. Importantly, this study is the first to describe the generation of a human Nrf2-null model, which is likely to have multiple applications in immunology and cancer biology. Collectively, this study demonstrates a role for Nrf2 in the effects of CDDO-Im on CD25 and IL-2 expression, whereas the effect of tBHQ on these parameters is complex and likely involves modulation of multiple stress-activated transcription factors, including NFκB and Nrf2.
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