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Page 708 of 1000-
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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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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Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.
The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific.
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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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Ultrasound-Mediated CRISPR Delivery and Sonogenetic Modulation for Precision Therapeutics.
The development of CRISPR-Cas9 technology has transformed gene editing with precise mutations in DNA targeted to treat genetic disorders yet various challenges such as immunogenicity, off-target editing, and lack of temporal control after delivery, making it difficult for therapeutic agents to reach deep tissues like brain, and cardiac tissues. Ultrasound has emerged as a promising solution by influencing sonogenetic modulation and delivery of CRISPR tools in deep tissues for targeted gene editing. Sonogenetic editing has revolutionized the technological advancement of CRISPR-editing and among multiple strategies, ultrasound-mediated delivery of CRISPR systems has enabled non-invasive and controlled genome editing through sonoporation, and upregulation of stimuli-responsive genes. Focused ultrasound combined with microbubbles has demonstrated CRISPR delivery to brain tissue with ∼80% higher efficiency than traditional strategies, allowing precise and advanced treatment of neurodegenerative diseases. In cancer applications, ultrasound has enabled complete tumor eradication via sonodynamic therapy, achieving 64% tumor regression via CAR T-cell therapy. Significant advances in gene therapy for cardiovascular diseases, obesity, and related diseases were also demonstrated recently. Collectively, the review elaborates potential of ultrasound-CRISPR technologies as a powerful tool that enables targeted, and image-guided genetic interventions, capable of advancing the outcomes of gene therapy as a widespread clinical practice.
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From the first human gene-editing to the birth of three-parent baby.
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Synergizing gene editing and cellular agriculture for a sustainable and healthy food future.
The intersection of gene editing and cellular agriculture is transforming food production by offering sustainable, ethical alternatives to conventional agriculture. Cellular agriculture uses tissue engineering and fermentation technologies to produce animal-free food, whereas gene editing tools like CRISPR-Cas9 optimize cellular efficiency, nutritional value, and sustainability. While some researchers emphasize the environmental and food security benefits, others raise concerns about high costs, regulatory challenges, and consumer acceptance. This paper critically examines existing literature, compares breakthroughs and controversies, and provides an expert perspective on the challenges and opportunities in gene-edited cellular agriculture. By tackling key scientific, economic, regulatory, and ethical issues, this article presents a roadmap for responsibly advancing these technologies and integrating them into global food systems. To our knowledge, this is the first work to explore how gene editing and cellular agriculture can be synergized to advance sustainability, food security, and global health.
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Prokaryotic Argonautes for in vivo biotechnology and molecular diagnostics.
Prokaryotic Argonautes (pAgos) are an emerging class of programmable endonucleases that are believed to be more flexible than existing CRISPR-Cas systems and have significant potential for biotechnology. Current applications of pAgos include a myriad of molecular diagnostics and in vitro DNA assembly tools. However, efforts have historically been centered on thermophilic pAgo variants. To enable in vivo biotechnological applications such as gene editing, focus has shifted to pAgos from mesophilic organisms. We discuss what is known of pAgos, how they are being developed for various applications, and strategies to overcome current challenges to in vivo applications in prokaryotes and eukaryotes.
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