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

Area of Science:

• Biotechnology
• Molecular Biology
• Genetic Engineering

Background:

• CRISPR-Cas9 technology enables precise genetic modification but faces challenges in efficient component delivery for therapeutic use.
• Developing effective delivery systems is crucial for advancing CRISPR-based therapies.

Purpose of the Study:

• To evaluate a modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) as a delivery vehicle for CRISPR-Cas9 ribonucleoprotein (RNP) complexes.
• To assess the efficiency and safety of the Ppoly system using the TILD-CRISPR method for gene integration.

Main Methods:

• Physicochemical properties, loading efficiency, and cellular uptake of RNP complexed with Ppoly were analyzed.
• TILD-CRISPR method was employed for GFP gene integration in CHO-K1 cells.
• Knock-in efficiency and cytotoxicity were compared between Ppoly and a commercial reagent (CRISPRMAX™).

Main Results:

• Ppoly demonstrated over 90% encapsulation efficiency for RNP with minimal cytotoxicity (cell viability >80%).
• The Ppoly-delivered TILD-CRISPR system achieved a 50% GFP gene integration efficiency in CHO-K1 cells.
• This significantly outperformed the 14% efficiency observed with CRISPRMAX™.

Conclusions:

• The cyclodextrin-based nanosponges (Ppoly) represent a promising platform for efficient and safe delivery of CRISPR-Cas9 components.
• This approach holds significant potential for applications in precise genome editing, therapeutic, and regenerative medicine.