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CRISPR-based technologies for cell biology.
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[The CRISPR/Cas system: a genome editing tool to develop animal models of viral infections].
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Gasdermin E as a potential target and biomarker for CRISPR-Cas9-based cancer therapy.
Gasdermin E (GSDME), a protein pivotal in mediating pyroptosis, has gained significant attention due to its role in cancer pathogenesis and its potential as a therapeutic target. The advent of CRISPR-Cas9, a precise genome editing tool, has revolutionized cancer therapy by enabling the manipulation of GSDME expression and function. This review explores the interplay of GSDME and CRISPR-Cas9 in cancer, emphasizing GSDME's unique mechanism of cleavage-dependent pore formation in the cell membrane and its emerging applications as both a therapeutic target and a diagnostic biomarker. We discuss the potential and challenges of using GSDME-induced pyroptosis as a therapeutic strategy and how can enhance its efficacy and specificity. We conclude by highlighting promising future research directions in this emerging field.
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Development of a gRNA Expression and Processing Platform for Efficient CRISPR-Cas9-Based Gene Editing and Gene Silencing in Candida tropicalis.
Candida tropicalis, a nonmodel diploid microbe, has been applied in industry as a chassis cell. Metabolic engineering of C. tropicalis is challenging due to a lack of gene editing and regulation tools. Here, we report a tRNA:guide RNA (gRNA) platform for boosting gene editing and silencing efficiency in C. tropicalis. As the endogenous tRNA-processing system enables autocleavage for producing a large number of mature gRNAs, a tRNAGly sequence from the genome of C. tropicalis ATCC 20336 was selected for constructing the tRNA:gRNA platform. In the CRISPR-Cas9 system, the tRNA:gRNA platform proved to be efficient in single-gene and multi-gene editing. Furthermore, based on the tRNA:gRNA platform, a CRISPR interference (CRISPRi) system was developed to construct an efficient dCas9-mediated gene expression regulation system for C. tropicalis. The CRISPRi system was employed to regulate the expression of the exogenous gene GFP3 (green fluorescent protein) and the endogenous gene ADE2 (phosphoribosylaminoimidazole carboxylase). Different regions of GFP3 and ADE2 were targeted with the gRNAs processed by the tRNAGly, and the transcription levels of GFP3 and ADE2 were successfully downregulated to 23.9% ± 4.1% and 38.0% ± 7.4%, respectively. The effects of the target regions on gene regulation were also investigated. Additionally, the regulation system was applied to silence ERG9 (squalene synthase) to enhance β-carotene biosynthesis in a metabolically modified C. tropicalis strain. The results suggest that the endogenous tRNAGly and the CRISPRi system have great potential for metabolic engineering of C. tropicalis. IMPORTANCE In the nonmodel yeast Candida tropicalis, a lack of available RNA polymerase type III (Pol III) promoters hindered the development of guide RNA (gRNA) expression platforms for the establishment of CRISPR-Cas-mediated genome editing and silencing strategies. Here, a tRNA:gRNA platform was constructed. We show that this platform allows efficient and precise expression and processing of different gRNAs from a single polycistronic gene capable of mediating multi-gene editing in combination with CRISPR-Cas9. Furthermore, in combination with dCas9, the tRNA:gRNA platform was efficiently used for silencing of exogenous and endogenous genes, representing the first CRISPR interference tool (CRISPRi) in C. tropicalis. Importantly, the established CRISPRi-tRNA:gRNA tool was also used for metabolic engineering by regulating β-carotene biosynthesis in C. tropicalis. The results suggest that the tRNA:gRNA platform and the CRISPRi system will further advance the application of the CRISPR-Cas-based editing and CRISPRi systems for metabolic engineering in C. tropicalis.
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Description of CRISPR/Cas9 development and its prospect in hepatocellular carcinoma treatment.
Hepatocellular carcinoma (HCC) is one of the most common malignancies today. Patients suffer from HCC since its high malignancy and limited treatment means. With the development of genetic research, new therapeutic strategy comes up in the way of gene editing. Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) was discovered as an immune sequence in bacteria and archaea. After artificial transformation and follow-up research, it is widely used as a gene editing tool. In this review, the development of CRISPR/Cas9 is summarized in retrospect. Through the evaluation of novel research in HCC, it is concluded that CRISPR/Cas9 would promote cancer research and provide a new tool for genetic treatment in prospect.
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Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.
CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster-derived peptide 'P2C' fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.
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Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters.
CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.
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CRISPR/Cas mediated epigenome editing for cancer therapy.
The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.
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The dCas9-SSAP as a promising genome editing tool in malaria parasites.
Measures to combat the Plasmodium parasites which cause malaria have become compromised because of reliance on a small arsenal of drugs, emerging drug resistance and the lack of effective vaccines. A promising avenue for addressing these challenges is the revolutionary gene-editing technology CRISPR-Cas9, due to its high efficiency and ease of design for genetic manipulation. The catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP)(dCas9-SSAP) is a recently emerged next-generation gene editing system added to the ever-growing CRISPR-Cas9-based technologies. While the classical Cas9-nuclease technologies are "double-strand break, damage-repair systems", the dCas9-SSAP is distinctively a "cleavage-free" editing tool. Unlimited to the Plasmodium genome, Cas9-nucleases imprint inheritable genetic scars on the subject genomes when applied. Here, we discussed the DSB genotoxicity pitfalls of existing nuclease-based editing tools, especially CRISPR-Cas9, and how the dCas9-SSAP presents a formidable option to the drawbacks within the context of Plasmodium genome editing. Then, we sought to infer a plausible mechanistic framework that could account for dCas9-SSAP-mediated genome editing. Finally, we discussed how dCas9-SSAP aligns with Plasmodium parasites' biology. This review would set the stage for continued research into the potential of this new, exciting technology in malaria parasites.
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Gene editing in liver diseases.
The deliberate and precise modification of the host genome using engineered nucleases represents a groundbreaking advancement in modern medicine. Several clinical trials employing these approaches to address metabolic liver disorders have been initiated, with recent remarkable outcomes observed in patients with transthyretin amyloidosis, highlighting the potential of these therapies. Recent technological improvements, particularly CRISPR Cas9-based technology, have revolutionized gene editing, enabling in vivo modification of the cellular genome for therapeutic purposes. These modifications include gene supplementation, correction, or silencing, offering a wide range of therapeutic possibilities. Moving forward, we anticipate witnessing the unfolding therapeutic potential of these strategies in the coming years. The aim of our review is to summarize preclinical data on gene editing in animal models of inherited liver diseases and the clinical data obtained thus far, emphasizing both therapeutic efficacy and potential limitations of these medical interventions.
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