Polyrotaxane-based multi-step transformable materials for the delivery of Cas9 ribonucleoprotein. (June 2022)

• Record Type: Journal Article
• Authors: Taharabaru, Toru; Kihara, Takuya; Onodera, Risako; Kogo, Tetsuya; Higashi, Kenjirou; Moribe, Kunikazu; Nakamura, Teruya; Motoyama, Keiichi; Higashi, Taishi
• Is Part Of: Applied materials today. Volume 27(2022)
• Publication Date: 2022-06
• Subjects: CRISPR-Cas9 -- Cas9 RNP -- Polyrotaxane -- Multi-step transformable supermolecules; Materials science -- Periodicals; Materials -- Research -- Periodicals; 620.1105
• DOI: 10.1016/j.apmt.2022.101488
• Languages: English
• ISSNs: 2352-9407

Abstract

Highlights: Multi-step transformable materials based on polyrotaxane (amino-PRXs) are developed Amino-PRXs allow efficient complexation with Cas9 RNP by simple mixing Multi-step transformable properties of amino-PRXs in the extra/intracellular environments are endowed by step-by-step structural optimization Fifth generation of amino-PRX is a safe, efficient, and convenient non-viral carrier for Cas9 RNP Abstract: 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 both in vitro and in vivo with significant usability, suggesting that amino-PRX (5 G) is a promising platform for the development of non-viral Cas9 RNP carriers. Graphical abstract: Image, graphical abstract