| Popis: |
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. |
