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Advancements in CRISPR/Cas systems for disease treatment.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is an adaptive immune system present in most bacteria and archaea, protecting them from infection by exogenous genetic elements. Due to its simplicity, cost-effectiveness, and precise gene editing capabilities, CRISPR/Cas technology has emerged as a promising tool for treating diseases. The continuous refinement of derivative systems has further broadened its scope in disease treatment. Nevertheless, the heterogeneous physiopathological nature of diseases and variations in disease onset sites pose significant challenges for in vivo applications of CRISPR systems. The efficiency of CRISPR systems in disease treatment is directly influenced by the performance of the delivery system. Additionally, concerns such as off-target effects present crucial hurdles in the clinical implementation of CRISPR systems. This review provides a comprehensive overview of the development of CRISPR systems, vector technologies, and their applications in disease treatment, while also addressing the challenges encountered in clinical settings. Furthermore, future research directions are outlined to pave the way for advancements in CRISPR-based therapies.
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A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.
CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.
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A Rapid and Efficient Method of Identifying G0 Males with Mosaic Germ Line Cells.
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Use the patent system to regulate gene editing.
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Programmable RNA-guided enzymes for next-generation genome editing.
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Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit.
The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering.
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Bypassing GMO regulations with CRISPR gene editing.
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The people behind the papers - Eduardo Leyva-Díaz and Oliver Hobert.
Transcriptional autoregulation occurs when transcription factors bind their own cis-regulatory sequences, ensuring their own continuous expression along with expression of other targets. During development, continued expression of identity-specifying transcription factors can be achieved by autoregulation, but until now formal evidence for a developmental requirement of autoregulation has been lacking. A new paper in Development provides this proof with the help of CRISPR/Cas9 gene editing in the C. elegans nervous system. We caught up with the paper's two authors: postdoc Eduardo Leyva-Díaz and his supervisor Oliver Hobert, Professor of Biological Sciences and HHMI Investigator at Columbia University, New York, to find out more about the work.
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Reducing CRISPR-Cas9 off-target effects by optically controlled chemical modifications of guide RNA.
A photocatalytic click chemistry approach, offering a significant advancement over conventional methods in RNA function modulation is described. This innovative method, utilizing light-activated small molecules, provides a high level of precision and control in RNA regulation, particularly effective in intricate cellular processes. By applying this strategy to CRISPR-Cas9 gene editing, we demonstrate its effectiveness in enhancing gene editing specificity and markedly reducing off-target effects. Our approach employs a vinyl ether modification in RNA, which activated under visible light with a phenanthrenequinone derivative, creating a CRISPR-OFF switch that precisely regulates CRISPR system activity. This method not only represents an advancement in genomic interventions but also offers broad applications in gene regulation, paving the way for safer and more reliable gene editing in therapeutic genomics.
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Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.
Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and β4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and β4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.
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