Abstract
The CRISPR-Cas9 system has quickly become the standard tool for genome editing. To deliver this system to target cells, adeno-associated virus (AAV) vectors are commonly used. In fact, AAV vectors have been utilized to deliver the CRISPR-Cas9 system to induce genomic exon skipping and restore the dystrophin protein in various Duchenne muscular dystrophy model animals. Despite the high transduction efficiency, AAV vector-mediated delivery has several limitations, such as the packaging size, prolonged overexpression of Cas9, immunogenicity against the AAV capsid, and the risk of integrating a part of the AAV genomic sequence into the host cell. To overcome these issues, we have recently engineered a transient delivery system utilizing VSV-G pseudotyped extracellular vesicles (EVs) termed NanoMEDIC (nanomembrane-derived extracellular vesicles for the delivery of macromolecular cargo). NanoMEDIC utilizes an HIV-derived Gag protein to package Cas9 protein and gRNA into EVs. The Cas9 and Gag proteins are fused to a heterodimerizer and conditionally dimerized by the addition of an inducible chemical ligand to recruit Cas9 protein into EVs. sgRNA is packaged into EVs through an HIV-derived RNA packaging signal and is subsequently released by two self-cleaving ribozymes. Utilizing these features, NanoMEDIC can achieve highly efficient packaging of the Cas9 protein and gRNA for genome editing into a variety of target cells and in vivo. Here, we describe a step-by-step protocol, including the gRNA-expressing vector construction and large-scale NanoMEDIC production, for in vivo genome editing.
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Acknowledgments
We are grateful to Dr. Yusuke Kojima, Dr. Peter Karagiannis, Dr. Eman Taha, and Joseph Lee for critical reading of the manuscript. This research was supported in part by AMED grants (JP19ek0109293, JP21bm0104001, JP21bm0804005, JP21im0210115), the T-CiRA Join Research program by Takeda Pharmaceutical Company, and an Intramural Research Grant (No. 28-6) for Neurological and Psychiatric Disorders of NCNP.
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Watanabe, K., Gee, P., Hotta, A. (2023). Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping. In: Maruyama, R., Yokota, T. (eds) Muscular Dystrophy Therapeutics. Methods in Molecular Biology, vol 2587. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2772-3_22
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DOI: https://doi.org/10.1007/978-1-0716-2772-3_22
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