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CRISPR/Cas9 genome editing in wheat.
Genome editing has been a long-term challenge for molecular biology research, particularly for plants possess complex genome. The recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile tool for genome editing which enables editing of multiple genes based on the guidance of small RNAs. Even though the efficiency of CRISPR/Cas9 system has been shown with several studies from diploid plants, its application remains a challenge for plants with polyploid and complex genome. Here, we applied CRISPR/Cas9 genome editing system in wheat protoplast to conduct the targeted editing of stress-responsive transcription factor genes, wheat dehydration responsive element binding protein 2 (TaDREB2) and wheat ethylene responsive factor 3 (TaERF3). Targeted genome editing of TaDREB2 and TaERF3 was achieved with transient expression of small guide RNA and Cas9 protein in wheat protoplast. The effectiveness of mutagenesis in wheat protoplast was confirmed with restriction enzyme digestion assay, T7 endonuclease assay, and sequencing. Furthermore, several off-target regions for designed sgRNAs were analyzed, and the specificity of genome editing was confirmed with amplicon sequencing. Overall results suggested that CRISPR/Cas9 genome editing system can easily be established on wheat protoplast and it has a huge potentiality for targeted manipulation of wheat genome for crop improvement purposes.
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CRISPR-Cas at a crossroads: from microbial immunity to precision biotechnology.
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) form RNA-guided adaptive immune systems in bacteria and archaea that mediate sequence-specific defense against invading genetic elements. Beyond their ecological role in restricting bacteriophage infection and horizontal gene transfer (HGT), CRISPR-Cas systems have been repurposed as programmable nucleases, enabling rapid, scalable, and precise genome engineering. Over the past decade, CRISPR platforms, most prominently Cas9, have transformed functional genomics, accelerated target discovery and drug development, and progressed from experimental tools to clinically evaluated gene and cell therapies. In parallel, growing attention has focused on both native and engineered roles of CRISPR-Cas in shaping HGT, plasmid ecology, and antimicrobial resistance (AMR), as AMR continues to expand globally. In this Review, we integrate advances spanning eukaryotic genome editing and prokaryotic antimicrobial applications. We summarize CRISPR-Cas classification and molecular mechanisms, highlighting spacer acquisition, guide RNA biogenesis, target recognition, and nucleic acid cleavage. We then examine how cellular DNA repair pathways influence editing outcomes and discuss strategies to enhance precision. We further review delivery strategies, such as conjugative plasmids, bacteriophages and phagemids, extracellular vesicles, and nanoparticles, together with evolutionary countermeasures encoded by mobile genetic elements, including anti-CRISPR proteins. Finally, we outline current limitations.
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Collateral damage and CRISPR genome editing.
The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.
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DNA-guided genome editing tool.
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Kinetics of Nuclear Uptake and Site-Specific DNA Cleavage during CRISPR-Directed Gene Editing in Solid Tumor Cells.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-directed gene editing is approaching clinical implementation in cancer. Thus, it is imperative to define the molecular framework upon which safe and efficacious therapeutic strategies can be built. Two important reaction parameters include the biological time frame within which the CRISPR/Cas complex enters the nucleus and executes gene editing, and the method of discrimination that the CRISPR/Cas complex utilizes to target tumor cell, but not normal cell, genomes. We are developing CRISPR-directed gene editing for the treatment of non-small cell lung carcinoma focusing on disabling Nuclear Factor Erythroid 2-Related Factor-Like (NRF2), a transcription factor that regulates chemoresistance and whose genetic disruption would enhance chemosensitivity. In this report, we define the time frame of cellular events that surround the initialization of CRISPR-directed gene editing as a function of the nuclear penetration and the execution of NRF2 gene disruption. We also identify a unique protospacer adjacent motif that facilitates site-specific cleavage of the NRF2 gene present only in tumor genomes. IMPLICATIONS: Our results begin to set a scientifically meritorious foundation for the exploitation of CRISPR-directed gene editing as an augmentative therapy for lung cancer and other solid tumors. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/6/891/F1.large.jpg.
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CRISPR-Cas engineering in food science and sustainable agriculture: recent advancements and applications.
The developments in the food supply chain to support the growing population of the world is one of today's most pressing issues, and to achieve this goal improvements should be performed in both crops and microbes. For this purpose, novel approaches such as genome editing (GE) methods have upgraded the biological sciences for genome manipulation and, among such methods, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are the main exciting innovations since the Green Revolution. CRISPR/Cas systems can be a potent tool for the food industry, improvement of agricultural crops and even for protecting food-grade bacteria from foreign genetic invasive elements. This review introduces the history and mechanism of the CRISPR-Cas system as a genome editing tool and its applications in the vaccination of starter cultures, production of antimicrobials and bioactive compounds, and genome editing of microorganisms.
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CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering.
The Chinese hamster ovary (CHO) cell line is a well-established platform for the production of biopharmaceuticals due to its ability to express complex therapeutic proteins with human-like glycopatterns in high amounts. The advent of CRISPR technology has opened up new avenues for the engineering of CHO cell lines for improved protein production and enhanced product quality. This review summarizes recent advances in the application of CRISPR technology for CHO cell line engineering with a particular focus on glycosylation modulation, productivity enhancement, tackling adventitious agents, elimination of problematic host cell proteins, development of antibiotic-free selection systems, site-specific transgene integration, and CRISPR-mediated gene activation and repression. The review highlights the potential of CRISPR technology in CHO cell line genome editing and epigenetic engineering for the more efficient and cost-effective development of biopharmaceuticals while ensuring the safety and quality of the final product.
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CRISPR/Cas technologies for cancer drug discovery and treatment.
Clustered regularly interspaced short palindromic repeats (CRISPR) tools are revolutionizing the establishment of genotype-phenotype relationships and are transforming cell- and gene-based therapies. In the field of oncology, CRISPR/CRISPR-associated protein 9 (Cas9), Cas12, and Cas13 have advanced the generation of cancer models, the study of tumor evolution, the identification of target genes involved in cancer growth, and the discovery of genes involved in chemosensitivity and resistance. Moreover, preclinical therapeutic strategies employing CRISPR/Cas have emerged. These include the generation of chimeric antigen receptor T (CAR-T) cells and engineered immune cells, and the use of precision anticancer gene-editing agents to inactivate driver oncogenes, suppress tumor support genes, and cull cancer cells in response to genetic circuit output. This review summarizes the collective impact that CRISPR technology has had on basic and applied cancer research, and highlights the promises and challenges facing its clinical translation.
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Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles.
Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.
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Research on genodermatoses using novel genome-editing tools.
Genodermatoses comprise a clinically heterogeneous group of mostly devastating disorders affecting the skin. To date, treatment options have in general been limited to symptom relief. However, the recent technical evolution in genome editing has ushered in a new era in the development of causal therapies for rare monogenetic diseases such as genodermatoses. The present review revisits the advantages and drawbacks of engineered nuclease tools currently available: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and - the most innovative - clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease 9 (CRISPR/Cas9) system. A mechanistic overview of the different modes of action of these programmable nucleases as well as their significance for causal therapy of genodermatoses is presented. Remaining limitations and challenges such as efficient delivery and off-target activity are critically discussed, highlighting both the past and future of gene therapy in dermatology.
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