Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration

Abstract

The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.