Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound-Propelled Nanomotors

Area of Science:

• Biotechnology
• Nanotechnology
• Molecular Biology

Background:

• Efficient intracellular delivery of gene editing tools like Cas9/sgRNA is crucial for therapeutic applications.
• Current methods face challenges in overcoming physiological barriers for effective delivery.

Purpose of the Study:

• To develop a rapid and direct intracellular delivery system for functional Cas9/sgRNA complexes.
• To utilize ultrasound-powered nanomotors for enhanced gene editing efficiency.

Main Methods:

• Cas9/sgRNA complex loaded onto nanomotor surfaces via disulfide linkage.
• Ultrasound treatment (5 min) to facilitate nanomotor penetration of cell membranes.
• Assessment of GFP gene knockout in GFP-expressing B16F10 cells.

Main Results:

• Nanomotors achieved direct plasma membrane penetration and intracellular release of Cas9/sgRNA.
• Over 80% GFP knockout achieved within 2 hours, significantly outperforming static nanowires (30%).
• High efficiency demonstrated with minimal Cas9/sgRNA complex (0.6 nm).

Conclusions:

• Ultrasound-powered nanomotors provide an effective strategy for direct intracellular delivery of functional proteins and RNAs.
• This nanomotor-based approach overcomes physiological barriers, showing significant promise for therapeutic applications.
• The method enables highly efficient gene knockout with reduced reagent usage.