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CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.
The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.
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History, evolution and classification of CRISPR-Cas associated systems.
This chapter provides a detailed description of the history of CRISPR-Cas and its evolution into one of the most efficient genome-editing strategies. The chapter begins by providing information on early findings that were critical in deciphering the role of CRISPR-Cas associated systems in prokaryotes. It then describes how CRISPR-Cas had been evolved into an efficient genome-editing strategy. In the subsequent section, latest developments in the genome-editing approaches based on CRISPR-Cas are discussed. The chapter ends with the recent classification and possible evolution of CRISPR-Cas systems.
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Geneticists retract study suggesting first CRISPR babies might die early.
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Novel CRISPR-Associated Gene-Editing Systems Discovered in Metagenomic Samples Enable Efficient and Specific Genome Engineering.
Development of medicines using gene editing has been hampered by enzymological and immunological impediments. We described previously the discovery and characterization of improved, novel gene-editing systems from metagenomic data. In this study, we substantially advance this work with three such gene-editing systems, demonstrating their utility for cell therapy development. All three systems are capable of reproducible, high-frequency gene editing in primary immune cells. In human T cells, disruption of the T cell receptor (TCR) alpha-chain was induced in >95% of cells, both paralogs of the TCR beta-chain in >90% of cells, and >90% knockout of β2-microglobulin, TIGIT, FAS, and PDCD1. Simultaneous double knockout of TRAC and TRBC was obtained at a frequency equal to that of the single edits. Gene editing with our systems had minimal effect on T cell viability. Furthermore, we integrate a chimeric antigen receptor (CAR) construct into TRAC (up to ∼60% of T cells), and demonstrate CAR expression and cytotoxicity. We next applied our novel gene-editing tools to natural killer (NK) cells, B cells, hematopoietic stem cells, and induced pluripotent stem cells, generating similarly efficient cell-engineering outcomes including the creation of active CAR-NK cells. Interrogation of our gene-editing systems' specificity reveals a profile comparable with or better than Cas9. Finally, our nucleases lack preexisting humoral and T cell-based immunity, consistent with their sourcing from nonhuman pathogens. In all, we show these new gene-editing systems have the activity, specificity, and translatability necessary for use in cell therapy development.
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Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.
Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.
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CRISPR-Cas9: a world first?
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Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine.
CRISPR/Cas9 is an effective gene editing tool with broad applications for the prevention or treatment of numerous diseases. It depends on CRISPR (clustered regularly interspaced short palindromic repeats) as a bacterial immune system and plays as a gene editing tool. Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing approaches, it has been broadly investigated to treat numerous hereditary and acquired illnesses, including cancers, hemolytic diseases, immunodeficiency disorders, cardiovascular diseases, visual maladies, neurodegenerative conditions, and a few X-linked disorders. CRISPR/Cas9 system has been used to treat cancers through a variety of approaches, with stable gene editing techniques. Here, the applications and clinical trials of CRISPR/Cas9 in various illnesses are described. Due to its high precision and efficiency, CRISPR/Cas9 strategies may treat gene-related illnesses by deleting, inserting, modifying, or blocking the expression of specific genes. The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed. The use of transfection vehicles for CRISPR/Cas9, including viral vectors (such as an Adeno-associated virus (AAV)), and the development of non-viral vectors is also considered.
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Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.
The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.
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Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.
We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.1.
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Efficient cancer modeling through CRISPR-Cas9/HDR-based somatic precision gene editing in mice.
CRISPR-Cas9 has been used successfully to introduce indels in somatic cells of rodents; however, precise editing of single nucleotides has been hampered by limitations of flexibility and efficiency. Here, we report technological modifications to the CRISPR-Cas9 vector system that now allows homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic tissues to generate tumor models with high flexibility and efficiency. Somatic editing of either Kras or Pik3ca in both normal and hyperplastic mammary glands led to swift tumorigenesis. The resulting tumors shared some histological, transcriptome, and proteome features with tumors induced by lentivirus-mediated expression of the respective oncogenes, but they also exhibited some distinct characteristics, particularly showing less intertumor variation, thus potentially offering more consistent models for cancer studies and therapeutic development. Therefore, this technological advance fills a critical gap between the power of CRISPR technology and high-fidelity mouse models for studying human tumor evolution and preclinical drug testing.
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