Polyrotaxane-based multi-step transformable materials for the delivery of Cas9 ribonucleoprotein
Polyrotaxane-based multi-step transformable materials for the delivery of Cas9 ribonucleoprotein
复制标题
DOI：
10.1016/j.apmt.2022.101488
复制
发表时间：
2022-06-01
影响因子：
8.3
通讯作者：
Higashi，Taishi
中科院分区：
文献类型：
--
作者：
Taharabaru，Toru;Kihara，Takuya;Higashi，Taishi
Cas9核糖核蛋白（Cas9 RNP）是一种有前途的基因组编辑工具，然而其生物学效用需要开发安全、有效和易于使用的非病毒载体。Cas9 RNP具有复杂的构象和电荷分布，导致与载体的低络合。此外，需要Cas9 RNP的细胞内摄取、内体逃逸、释放和核转位。在这里，我们报告了基于聚轮烷的超分子载体，胺化聚轮烷（氨基-PRX）的开发，其通过其自主转化特性（第1代; 1G）与Cas9 RNP有效地形成复合物。此外，氨基-PRX的氨基被优化以通过在内体中转化为高度阳离子颗粒来提供内体逃逸能力（2G）。此外，为Cas9 RNP释放（3G-5G）提供了细胞内降解性质，导致释放的Cas9 RNP定位于细胞核中。最后，我们证明了这种优化的amino-PRX（5G）促进了体外和体内的高效基因组编辑，具有显著的可用性，表明amino-PRX（5G）是开发非病毒Cas9 RNP载体的有希望的平台。
Cas9 ribonucleoprotein (Cas9 RNP) is a promising genome editing tool, however its biological utility reuires the development of safe, efficient, and easy-to-use non-viral carriers. Cas9 RNP has a complicated conformation and charge distribution, resulting in low complexation with carriers. In addition, intracellular uptake, endosome escape, release, and nuclear translocation of Cas9 RNP are required. Here, we report the development of polyrotaxane-based supramolecular carriers, aminated polyrotaxanes (amino-PRXs), that efficiently form complexes with Cas9 RNP via their autonomous transforming properties (1st generation; 1 G). Further, the amino groups of amino-PRXs are optimized to provide endosome-escape ability (2 G) via transforming to highly cationic particles in the endosome. Moreover, intracellular degradation properties are provided for Cas9 RNP release (3G–5 G) resulting in released Cas9 RNP becoming localized in the nucleus. Finally, we demonstrate that this optimized amino-PRX (5 G) facilitates highly efficient genome editing bothin vitroandin vivowith significant usability, suggesting that amino-PRX (5 G) is a promising platform for the development of non-viral Cas9 RNP carriers.