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

Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe₃O₄ nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe₃O₄ nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.