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CRISPR: MODIFYING THE LIFE SCIENCE LANDSCAPE.
From lampreys to human stem cells, the CRISPR/Cas9 system is challenging our notions of what is possible with genome editing. Nathan Blow talks to researchers pushing the boundaries of CRISPR/Cas9 technology to expand our understanding of biology.
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Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line.
INTRODUCTION: Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. MATERIALS AND METHODS: Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. RESULTS: Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. CONCLUSIONS: Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.
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Gene Editing by Extracellular Vesicles.
CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.
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CRISPR/Cas9 gene editing in muscle-related genetic disorders: Restoring function and exercise capacity.
Due to their progressive and debilitating character and lack of curative therapies, muscle-related hereditary illnesses such as muscular dystrophies (MDs), myopathies, and motor neuron diseases need immediate care. A pressing need exists for novel therapeutics that can accurately and persistently correct the underlying mutations in muscle-related genetic disorders, as existing treatments are mostly palliative rather than addressing the underlying genetic cause. Traditional therapies for genetic diseases, such as hereditary myopathies and MDs, are often insufficient; however, the advent of CRISPR/Cas9 technology has altered this trajectory. While other evaluations have compiled the potential of gene editing, this one compiles the latest findings on CRISPR/Cas9 applications for musculoskeletal disorders. With an emphasis on the repair of recessive and dominant-negative mutations, we provide a critical evaluation of the development of new therapeutic vectors and in vivo editing techniques that have gone from conceptual models to preclinical triumphs. More specifically, we address the issues of efficient delivery, off-target effects, and immunological responses specific to muscle tissue, all of which are ongoing challenges. By facilitating targeted correction of pathogenic mutations, CRISPR/Cas9 holds promise for restoring muscle integrity and functional performance, potentially translating into improved exercise capacity. This study offers a prospective view on the near-term therapeutic promise of CRISPR/Cas9 for treating crippling muscle diseases by combining recent advances with an evaluation of the translational route.
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A fast method to reprogram and CRISPR/Cas9 gene editing from erythroblasts.
An efficient one-step procedure to reprogram fibroblasts into human induced pluripotent stem cells (hiPSC) and perform CRISPR/Cas9 gene editing simultaneously was recently reported. Here we show that such simultaneous reprogramming and gene editing can be efficiently done from erythroblasts. We successfully obtained human induced pluripotent stem cells colonies together with in frame and out of frame CAPN1 mutations in one or both alleles. We did not identify off-targets in edited cell lines. The entire process, from blood collection to mutated hiPSC took approximately 5 weeks, a much shorter period than standard multi-step methodologies using fibroblasts. Noteworthy, blood drawing is a less invasive procedure than a skin biopsy.
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Extreme Genome Editing.
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CRISPR/CAS9 Technologies.
The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) pathway is revolutionizing biological research. Modifications to this primitive prokaryotic immune system now enable scientists to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms. Thus, many laboratories can now perform important experiments that previously were considered scientifically risky or too costly. Here, we describe the components of the CRISPR/Cas system that have been engineered for use in eukaryotes. We also explain how this system can be used to genetically modify cell lines and model organisms, or regulate gene expression in order to search for new participants in biological pathways. © 2017 American Society for Bone and Mineral Research.
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Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies - a spotlight on hemophilia.
INTRODUCTION: Currently, a few in vivo gene replacement therapies are commercially available, with many in clinical development for the treatment of some inherited monogenic diseases. These disorders arise from mutations in genes encoding essential proteins with a well understood biological function. Wide adoption of gene replacement therapies requires solid safety and efficacy profiles with demonstrable long-term durability and cost-benefit advantages vs standard therapies. AREAS COVERED: This expert review outlines the challenges and opportunities in treating hemophilia, including the progression from in vivo gene therapies toward in vivo gene editing, focusing on pre-clinical and emerging clinical data for gene editing and addressing the need for sustained and durable gene expression during hepatocyte proliferation when the liver is unable to maintain steady gene expression and protein production. EXPERT OPINION: In vivo gene editing in liver tissues may be able to rescue patients younger than 18 years who are not eligible for gene replacement therapies, with hemophilia as a prime example.
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Creation of a Novel Humanized Dystrophic Mouse Model of Duchenne Muscular Dystrophy and Application of a CRISPR/Cas9 Gene Editing Therapy.
Duchenne muscular dystrophy is caused by mutations in DMD which disrupt the reading frame. Therapeutic strategies that restore DMD's reading frame, such as exon skipping and CRISPR/Cas9, need to be tested in the context of the human DMD sequence in vivo. We have developed a novel dystrophic mouse model by using CRISPR/Cas9 to delete exon 45 in the human DMD gene in hDMD mice, which places DMD out-of-frame. We have utilized this model to demonstrate that our clinically-relevant CRISPR/Cas9 platform, which targets deletion of human DMD exons 45-55, can be directly applied in vivo to restore dystrophin.
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Advances in Engineering the Fly Genome with the CRISPR-Cas System.
Drosophila has long been a premier model for the development and application of cutting-edge genetic approaches. The CRISPR-Cas system now adds the ability to manipulate the genome with ease and precision, providing a rich toolbox to interrogate relationships between genotype and phenotype, to delineate and visualize how the genome is organized, to illuminate and manipulate RNA, and to pioneer new gene drive technologies. Myriad transformative approaches have already originated from the CRISPR-Cas system, which will likely continue to spark the creation of tools with diverse applications. Here, we provide an overview of how CRISPR-Cas gene editing has revolutionized genetic analysis in Drosophila and highlight key areas for future advances.
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