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Developing Methods to Circumvent the Conundrum of Chromosomal Rearrangements Occurring in Multiplex Gene Edition.
CRISPR/Cas9 is a powerful tool to edit the genome of the yeast Yarrowia lipolytica. Here, we design a simple and robust method to knockout multiple gene families based on the construction of plasmids enabling the simultaneous expression of several sgRNAs. We exemplify the potency of this approach by targeting the well-characterized acyl-CoA oxidase family (POX) and the uncharacterized SPS19 family. We establish a correlation between the high lethality observed upon editing multiple loci and chromosomal translocations resulting from the simultaneous generation of several double-strand breaks (DSBs) and develop multiplex gene editing strategies. Using homologous directed recombination to reduce chromosomal translocations, we demonstrated that simultaneous editing of four genes can be achieved and constructed a strain carrying a sextuple deletion of POX genes. We explore an "excision approach" by simultaneously performing two DSBs in genes and reached 73 to 100% editing efficiency in double disruptions and 41.7% in a triple disruption. This work led to identifying SPS193 as a gene encoding a 2-4 dienoyl-CoA reductase, demonstrating the potential of this method to accelerate knowledge on gene function in expanded gene families.
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Optimization of CRISPR/Cas9-based multi-gene editing system in Aspergillus oryzae and its application in α-amyrin biosynthesis.
Aspergillus oryzae, with its high protein secretion capacity, post-translational modification capabilities, and safety, is a promising host for producing natural products and recombinant proteins. However, the lack of efficient genetic tools and precise genome modification methods has significantly slowed progress in the metabolic engineering of A. oryzae. Although the CRISPR/Cas9-mediated genome editing system has been applied in A. oryzae, the low efficiency of gene disruption and heterologous gene integration limits its widespread application as a chassis cell for industrial strain development. In this study, the CRISPR/Cas9-mediated genome editing system in A. oryzae RIB40 was optimized to significantly improve its editing efficiency. By evaluating the effect of promoters on sgRNA expression, it was determined that the Ao (Up338)5SrRNA promoter effectively enhances the gene disruption efficiency. Utilizing the Ao (Up338)5SrRNA promoter, a multiplex gene editing system based on the tRNAGly-sgRNA array was developed, achieving dual-gene disruption efficiency of 78.84% and triple-gene disruption efficiency of 41.15%. Additionally, when the ratio of the genome-editing plasmid to the circular donor DNA was 1:3, the site-specific integration efficiency of the exogenous gene reached 61.36%. Based on the developed CRISPR/Cas9 genome editing system, the heterologous α-amyrin synthetic gene (CADDS) was precisely integrated into the wA locus to achieving the biosynthesis of α-amyrin in Aspergillus oryzae. Furthermore, acetyl-CoA supply was improved by knocking out the competing ethanol metabolic pathway, resulting in an engineered strain that produced 0.69 mg/g of α-amyrin.
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Recent advances of CRISPR-based gene editing technologies and delivery strategies.
CRISPR technology is a powerful tool for gene editing, in which the efficient delivery of living target cells allows it to show great clinical potential. At present, the commonly used in vivo delivery strategies mainly include biological methods (AAV, VLP, SEND) and chemical methods (LNP), which subtly deliver gene editors to living target cells safely and efficiently from different ways. However, existing delivery systems have different extents of limitations in terms of editing efficiency, immunogenicity, half-life, etc., so developing optimized delivery systems is the key to fully realizing the potential of CRISPR-Cas system for intracellular gene editing. In order to fully understand the advantages of different delivery strategies to maximize the ability to help CRISPR systems choose delivery methods, we conducted a systematic review. In this paper, we introduce the types, principles and characteristics of gene editing systems in order to understand their requirements for delivery tools. We focus on describing the type, principle, load, immunogenicity, specificity, toxicity, etc. of the delivery system, so as to fully analyse its advantages and disadvantages for the selection of different editing environments. This review aims to provide new insights to facilitate appropriate delivery systems or improve the efficacy of existing systems.
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Genome Editing of Induced Pluripotent Stem Cells Using CRISPR/Cas9 Ribonucleoprotein Complexes to Model Genetic Ocular Diseases.
Genome editing with the use of CRISPR/Cas9 ribonucleoprotein complexes of induced pluripotent stem cells can be used to model many diseases. The combination of stem cells and gene editing technologies is a valuable tool to study ocular disorders, as many have been identified to be caused by specific genetic mutations. This protocol provides experimentally derived guidelines for genome editing of human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 ribonucleoprotein complexes to generate iPSCs harboring single nucleotide variants from ocular disorders. Edited iPSC can be further differentiated into retinal cells in order to study disease mechanisms as well as screen potential therapies.
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CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.
DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.
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The genome-editing decade.
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Gene Knockout in Leptospira spp. by CRISPR/Cas9-NHEJ (Non-Homologous End-Joining) and CRISPR-Prime Editing.
Genetic manipulation of Leptospira spp. has progressed significantly in recent years. Like most prokaryotes, leptospires are unable to survive double-strand breaks (DSBs) induced by the Cas9 endonuclease, prompting the development of alternative strategies for gene knockout. We have established two systems for targeted mutagenesis of Leptospira spp.: CRISPR/Cas9-NHEJ and CRISPR-Prime Editing (PE). The CRISPR/Cas9-NHEJ approach involves coexpression of the CRISPR/Cas9 machinery alongside the DNA repair proteins LigD and Ku from Mycobacterium smegmatis, facilitating error-prone repair of DSBs that results in indel mutations. In contrast, CRISPR-PE is a DSB-free strategy that utilizes a Cas9-nickase fused to a reverse transcriptase that facilitates precise single-nucleotide edits in the genome. This expanded toolbox has placed Leptospira spp. at the forefront of bacterial genetic manipulation.
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Editing the immune system in vivo in mice using CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing of transplanted hematopoietic stem cells.
CRISPR/Cas9-based genome editing has been widely used to evaluate target gene function in biomedical research. The CRISPR/Cas9 system can introduce gene knockout, knock-in and mutations with more ease than earlier generations of genome editing tools. Using CRISPR/Cas9-based genome editing, researchers have successfully modified the DNA of different immune components, including primary T cells, B cells, macrophages, and immune system progenitors, i.e. hematopoietic stem cells (HSCs), which are also known as Lin-Sca1 + Kit + cells (LSKs) in mice. We previously reported that the transplantation of HSCs with lentivirus-mediated CRISPR/Cas9-based genetic modifications into lethally irradiated congenic mice repopulated the ablated recipient immune system with the donor immune system. In this report, we efficiently manipulated CD40 expression in LSK cells using Cas9 RNP and demonstrated the functional impact in a colitis model. Compared to a virus-based strategy, the RNP approach has the potential to enable investigation of target gene biology in any mouse strain and eliminates the time and effort associated with virus production and infection. Therefore, in vivo RNP-based CRISPR/Cas9 gene editing of transplanted HSCs represents a promising new strategy for exploring gene function in the immune system of mice.
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[The prospects of using the technology of genome editing for correction of neurodegenerative diseases.].
We reviewed the results of modern studies using gene therapy methods for correction of neurodegenerative diseases. We analyzed approaches based on the technologies of gene editing and their prospective use in medicine. We discussed some limitations associated with the features of pathogenesis of neurodegenerative diseases for application of the genome editing tools directing to for the treatment of these pathologies.
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Application of CRISPR-Cas9 based gene editing to study the pathogenesis of colon and liver cancer using organoids.
Two breakthrough techniques that have totally revolutionized biology in last 1 decade are the discovery of genome editing tools and growing the stem cells/primary tissue explants in defined 3D culture. In this regard the discovery of CRISPR-Cas9 as a specific gene editing tool and organoid culture from adult stem cell has provided easy handy tools to uncover the process of organ development and also modeling cancer. Genetically modified organoids have been developed by sequential knockout and knockin of driver mutations by genome editing followed by niche-based selection. The modified organoids when xenotransplanted in animal models faithfully recapitulate the neoplastic events of human tumors. The present review focuses on the merging of these two powerful technologies in understanding the complexities of colon and liver cancer.
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