Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing

Area of Science:

• Biomedical Engineering
• Nanotechnology
• Gene Therapy

Background:

• Intracellular delivery of CRISPR/Cas9 gene-editing tools is hindered by cytoplasmic viscosity and lysosomal entrapment.
• Efficient cytosolic transport is crucial for effective gene editing and therapeutic applications.

Purpose of the Study:

• To develop magnetically actuated nanorobots (MagCbots) for enhanced intracellular delivery of CRISPR/Cas9.
• To investigate the ability of MagCbots to navigate viscous intracellular environments and improve gene-editing efficiency.

Main Methods:

• Fabrication of magnetic nanoparticle clusters (Fe3O4) with tunable size and magnetic responsiveness.
• Assembly of MagCbots (approx. 200 nm) by electrostatic interaction between Fe3O4 nanoclusters and CRISPR-Cas9 plasmids.
• Magnetic actuation to propel MagCbots within the cytoplasm and assess intracellular viscosity reduction and lysosomal escape.

Main Results:

• MagCbots achieved propulsion (0.41 μm/s) in viscous environments, reducing viscosity by ~50% and increasing lysosomal escape 3-fold.
• The nanorobot's porous structure provided high payload capacity and protected plasmid DNA from degradation.
• Efficient genome editing of PD1 and PLK1 genes was demonstrated in various cell lines, including difficult-to-transfect Jurkat T cells.

Conclusions:

• Magnetically driven MagCbots offer an effective solution for active intracellular delivery of gene-editing components.
• This platform shows significant potential for advancing gene therapy and other biomedical applications requiring precise intracellular manipulation.