Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.
MaxCyte Inc., Gaithersburg, MD, USA.
Methods Mol Biol. 2023;2587:427-453. doi: 10.1007/978-1-0716-2772-3_22.
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.
CRISPR-Cas9 系统已迅速成为基因组编辑的标准工具。为了将该系统递送至靶细胞，常使用腺相关病毒（AAV）载体。事实上，已利用 AAV 载体将 CRISPR-Cas9 系统递送至各种杜氏肌营养不良症模型动物以诱导基因组外显子跳跃并恢复肌营养不良蛋白。尽管 AAV 载体介导的递送具有很高的转导效率，但存在几个局限性，如包装大小、Cas9 的延长过表达、针对 AAV 衣壳的免疫原性以及 AAV 基因组序列的一部分整合到宿主细胞的风险。为了克服这些问题，我们最近设计了一种利用 VSV-G 假型化细胞外囊泡（EVs）的瞬时递送系统，称为 NanoMEDIC（用于递送大分子货物的纳米膜衍生细胞外囊泡）。NanoMEDIC 利用 HIV 衍生的 Gag 蛋白将 Cas9 蛋白和 gRNA 包装到 EVs 中。Cas9 和 Gag 蛋白融合到异二聚体中，并通过添加诱导化学配体条件性二聚化，将 Cas9 蛋白募集到 EVs 中。sgRNA 通过 HIV 衍生的 RNA 包装信号包装到 EVs 中，并随后通过两个自我切割核酶释放。利用这些特性，NanoMEDIC 可以高效地将 Cas9 蛋白和 gRNA 包装到各种靶细胞和体内进行基因组编辑。在此，我们描述了一种逐步方案，包括 gRNA 表达载体的构建和大规模 NanoMEDIC 的生产，用于体内基因组编辑。
