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Found: 26 139
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Targeting genomic rearrangements in tumor cells through Cas9-mediated insertion of a suicide gene.
Specifically targeting genomic rearrangements and mutations in tumor cells remains an elusive goal in cancer therapy. Here, we used Cas9-based genome editing to introduce the gene encoding the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of cancer cells carrying unique sequences resulting from genome rearrangements. Specifically, we targeted the breakpoints of TMEM135-CCDC67 and MAN2A1-FER fusions in human prostate cancer or hepatocellular carcinoma cells in vitro and in mouse xenografts. We designed one adenovirus to deliver the nickase Cas9D10A and guide RNAs targeting the breakpoint sequences, and another to deliver an EGFP-HSV1-tk construct flanked by sequences homologous to those surrounding the breakpoint. Infection with both viruses resulted in breakpoint-dependent expression of EGFP-tk and ganciclovir-mediated apoptosis. When mouse xenografts were treated with adenoviruses and ganciclovir, all animals showed decreased tumor burden and no mortality during the study. Thus, Cas9-mediated suicide-gene insertion may be a viable genotype-specific cancer therapy.
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NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans.
GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7 ) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.
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CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.
RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.
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Extracellular vesicle and CRISPR gene therapy: Current applications in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease.
Neurodegenerative diseases are characterized by progressive deterioration of the nervous system. Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD) are prominently life-threatening examples of neurodegenerative diseases. The complexity of the pathophysiology in neurodegenerative diseases causes difficulties in diagnosing. Although the drugs temporarily help to correct specific symptoms including memory loss and degeneration, a complete treatment has not been found yet. New therapeutic approaches have been developed to understand and treat the underlying pathogenesis of neurodegenerative diseases. With this purpose, clustered-regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has recently suggested a new treatment option. Editing of the genome is carried out by insertion and deletion processes on DNA. Safe delivery of the CRISPR/Cas system to the targeted cells without affecting surrounding cells is frequently investigated. Extracellular vesicles (EVs), that is exosomes, have recently been used in CRISPR/Cas studies. In this review, CRISPR/Cas and EV approaches used for diagnosis and/or treatment in AD, PD, ALS, and HD are reviewed. CRISPR/Cas and EV technologies, which stand out as new therapeutic approaches, may offer a definitive treatment option in neurodegenerative diseases.
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[Research progress of the CRISPR-Cas system in the detection and elimination of antibiotic resistance genes].
The abuse of antibiotics has led to increasingly severe antibiotic resistance, which poses a serious threat to global public health security. Therefore, the rapid detection and effective elimination of antibiotic resistance genes are crucial for controlling the spread of drug-resistant bacteria. Conventional methods for detecting antibiotic resistance genes, such as PCR, quantitative real-time PCR, and isothermal amplification, have shortcomings including time-consuming procedures, labor intensiveness, high costs, and poor specificity. Therefore, there is an urgent need to find new solutions to combat bacterial resistance, making the development of novel and efficient technologies for detecting and eliminating antibiotic resistance genes. In recent years, gene editing technology has become a research hotspot. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins) consisted of the CRISPR-Cas system. As one of the adaptive immune systems in prokaryotes, it possesses unique sequence targeting ability and high sensitivity, which enable it to accurately recognize and cleave target nucleic acid sequences. Thus, it shows great potential in the detection and elimination of antibiotic resistance genes. This review summarizes the classification, structures, and action mechanisms of CRISPR-Cas systems, elaborates on the application of different CRISPR-Cas systems in the detection and elimination of antibiotic resistance genes, and finally discusses the challenges faced by this technology and its future development directions, aiming to provide new ideas for addressing antibiotic resistance in pathogenic bacteria.
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Characterization and phosphoproteomic analysis of a human immortalized podocyte model of Fabry disease generated using CRISPR/Cas9 technology.
Fabry nephropathy is a major cause of morbidity and premature death in patients with Fabry disease (FD), a rare X-linked lysosomal storage disorder. Gb3, the main substrate of α-galactosidase A (α-Gal A), progressively accumulates within cells in a variety of tissues. Establishment of cell models has been useful as a tool for testing hypotheses of disease pathogenesis. We applied CRISPR/Cas9 genome editing technology to the GLA gene to develop human kidney cell models of FD in human immortalized podocytes, which are the main affected renal cell type. Our podocytes lack detectable α-Gal A activity and have increased levels of Gb3. To explore different pathways that could have distinct patterns of activation under conditions of α-gal A deficiency, we used a high-throughput antibody array to perform phosphorylation profiling of CRISPR/Cas9-edited and control podocytes. Changes in both total protein levels and in phosphorylation status per site were observed. Analysis of our candidate proteins suggests that multiple signaling pathways are impaired in FD.
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High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus.
In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran Oncopeltus fasciatus The white gene is well characterized in Drosophila where mutation causes loss of eye pigmentation; white is a reliable marker for transgenesis and other genetic manipulations. Accordingly, white has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the Of-white (Of-w) locus using CRISPR/Cas9. We found that Of-w is required for pigmentation throughout the body of Oncopeltus, not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, Of-w mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied Of-w homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in Oncopeltus While we were unable to establish white as a useful visible marker for Oncopeltus, these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic Drosophila gene.
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Engineered Extracellular Vesicle-Delivered CRISPR/CasRx as a Novel RNA Editing Tool.
Engineered extracellular vesicles (EVs) are considered excellent delivery vehicles for a variety of therapeutic agents, including nucleic acids, proteins, drugs, and nanomaterials. Recently, several studies have indicated that clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) delivered by EVs enable efficient DNA editing. However, an RNA editing tool delivered by EVs is still unavailable. Here, a signal peptide-optimized and EVs-delivered guide RNA (gRNA) and CRISPR/CasRx (Cas13d) system capable of rapidly inhibiting the expression of targeted genes with quick catabolism after performing their functions is developed. EVs with CRISPR/CasRx and tandem gRNAs targeting pivotal cytokines are further packed whose levels increase substantially over the course of acute inflammatory diseases and find that these engineered EVs inhibit macrophage activation in vitro. More importantly, this system attenuates lipopolysaccharide (LPS)-triggered acute lung injury and sepsis in the acute phase, mitigating organ damage and improving the prognosis in vivo. In summary, a potent tool is provided for short-acting RNA editing, which could be a powerful therapeutic platform for the treatment of acute diseases.
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CRISPR/Cas9-mediated gene deletion of the ompA gene in symbiotic Cedecea neteri impairs biofilm formation and reduces gut colonization of Aedes aegypti mosquitoes.
BACKGROUND: Symbiotic bacteria are pervasive in mosquitoes and their presence can influence many host phenotypes that affect vectoral capacity. While it is evident that environmental and host genetic factors contribute in shaping the microbiome of mosquitoes, we have a poor understanding regarding how bacterial genetics affects colonization of the mosquito gut. The CRISPR/Cas9 gene editing system is a powerful tool to alter bacterial genomes facilitating investigations into host-microbe interactions but has yet to be applied to insect symbionts. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of bacterial genetic factors in mosquito biology and in colonization of mosquitoes we used CRISPR/Cas9 gene editing system to mutate the outer membrane protein A (ompA) gene of a Cedecea neteri symbiont isolated from Aedes mosquitoes. The ompA mutant had an impaired ability to form biofilms and poorly infected Ae. aegypti when reared in a mono-association under gnotobiotic conditions. In adult mosquitoes, the mutant had a significantly reduced infection prevalence compared to the wild type or complement strains, while no differences in prevalence were seen in larvae, suggesting genetic factors are particularly important for adult gut colonization. We also used the CRISPR/Cas9 system to integrate genes (antibiotic resistance and fluorescent markers) into the symbionts genome and demonstrated that these genes were functional in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: Our results shed insights into the role of ompA gene in host-microbe interactions in Ae. aegypti and confirm that CRISPR/Cas9 gene editing can be employed for genetic manipulation of non-model gut microbes. The ability to use this technology for site-specific integration of genes into the symbiont will facilitate the development of paratransgenic control strategies to interfere with arboviral pathogens such Chikungunya, dengue, Zika and Yellow fever viruses transmitted by Aedes mosquitoes.
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Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR.
Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G2 cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.
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