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HPV-driven cancers: a looming threat and the potential of CRISPR/Cas9 for targeted therapy.
Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.
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Quantification of Genome Editing and Transcriptional Control Capabilities Reveals Hierarchies among Diverse CRISPR/Cas Systems in Human Cells.
CRISPR/Cas technologies have revolutionized the ability to redesign genomic information and tailor endogenous gene expression. Nevertheless, the discovery and development of new CRISPR/Cas systems has resulted in a lack of clarity surrounding the relative efficacies among these technologies in human cells. This deficit makes the optimal selection of CRISPR/Cas technologies in human cells unnecessarily challenging, which in turn hampers their adoption, and thus ultimately limits their utility. Here, we designed a series of endogenous testbed systems to methodically quantify and compare the genome editing, CRISPRi, and CRISPRa capabilities among 10 different natural and engineered Cas protein variants spanning Type II and Type V CRISPR/Cas families. We show that although all Cas protein variants are capable of genome editing and transcriptional control in human cells, hierarchies exist, particularly for genome editing and CRISPRa applications, wherein Cas9 ≥ Cas12a > Cas12e/Cas12j. Our findings also highlight the utility of our modular testbed platforms to rapidly and systematically quantify the functionality of practically any natural or engineered genomic-targeting Cas protein in human cells.
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Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.
OBJECTIVES: To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins. RESULTS: To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations. CONCLUSIONS: We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.
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Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal.
Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.
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Application of Gene Therapy in Hemophilia.
Gene therapy refers to introducing normal exogenous genes into target cells to correct or compensate for the diseases caused by defective and abnormal genes for the purpose of therapy. It holds out hope of a cure for single-gene genetic diseases such as thalassemia, hemophilia, etc. At present, gene therapy is performed in two ways: introducing exogenous genes, and gene editing. A great number of clinical trials of gene therapy in hemophilia have been carried out using viral vectors to introduce foreign genes into target cells. However, the production of neutralizing antibodies following injection and the inability to prepare viral vectors in large quantities limit their application. Although gene-editing methods like CRISPR avoid the above problems, the potential risks of off-target effects are still unknown. More trials and evidence are needed to elucidate the safety and accuracy of gene therapy. This paper will review the bench and clinical work of gene therapy in hemophilia in recent years, and summarize the challenges and prospects of gene therapy, so as to provide directions for future scientific research in this field.
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Development of Inorganic and Hybrid Nanosystems for Delivery of CRISPR-Based Gene Editors.
The CRISPR-Cas9 system is a gene editing tool, replacing specific target sequences of eukaryotic DNA via a distinct molecular pathway, with minimal off-target effects. In this manner, the effects are long-lasting and potentially require only one dose of medication to be effective. The package of bacterial nucleases is susceptible to the same degradative pathways as other nucleic acid therapeutics. Similarly, the CRISPR-Cas9 system is incapable of traversing biological membranes on its own. Therefore, a delivery mechanism is needed for effective transfection. In recent literature reviews, the focus has been on viral and lipid-based drug delivery systems, with little attention paid to solid-core nanoparticles, such as gold or silica nanoparticles, which present unique physicochemical properties and delivery opportunities. While viral delivery systems are efficient carriers, they can be highly immunogenic and unstable, and the production of high-viral titers is limited. Lipid-based drug delivery systems, such as liposomes, possess good shelf life, encapsulation, and transfection efficiency, but their biological stability and biodistribution profile limit their in vivo use. Thus, the arsenal of delivery agents could be complemented by solid-core nanoparticles. Their unique structural properties could lead to improved delivery strategies to mitigate disease outcomes and promote organ-specific delivery.
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Membrane-destabilizing ionizable phospholipids: Novel components for organ-selective mRNA delivery and CRISPR-Cas gene editing.
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CRISPR is so popular even viruses may use it.
Thousands of phages appear to have stolen the gene-cutting mechanism.
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Approaches for CRISPR/Cas9 Genome Editing in C. elegans.
The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe our most up-to-date protocols using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. We present several considerations when deciding which strategy best suits the needs of the experiment.
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Generating mammalian knock-out cell lines to investigate mitochondrial protein complex assembly.
The development of easy-to-use gene-editing approaches has revolutionized the study of mitochondrial protein complex assembly in mammalian cells. Once the domain of classical cell biology models such Saccharomyces cerevisiae, human knockout cell lines lacking expression of a specific protein can be made at low cost and in as little as two to three weeks. In this chapter we outline our approach to generation of knockouts in commonly used transformed laboratory cell lines, with a view toward their use in the study of mitochondrial respiratory chain complex assembly and mitochondrial biology. Common pitfalls and caveats are discussed along with recommendations on how to validate a knockout cell line through sequencing of genomic edits and stable complementation to exclude the influence of off-target effects and enable further studies of protein function.
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