The advent of RNA-guided endonucleases has undeniably transformed gene editing and biotechnology. The CRISPR/Cas9 system, known for its precision and simplicity, enables targeted genome editing without complex protein engineering. However, traditional plasmid transfection of Cas9 and guide RNA can lead to unwanted permanent genetic modifications and immune responses. A more advantageous approach is the direct delivery of transient Cas9 ribonucleoprotein, offering significant potential for gene editing and therapeutic applications of CRISPR/Cas9.
Efficient gene editing using covalent fusion of Cas9 with cell-penetrating peptides often requires lengthy incubation periods. Furthermore, recent studies suggest that conjugating the anionic Cas9 ribonucleoprotein to cationic peptides via covalent bonds may hinder nuclease activity due to unfavorable electrostatic interactions.
Our research introduces an innovative supramolecular strategy for direct Cas9 delivery, employing an amphiphilic penetrating peptide. This peptide was synthesized through a hydrazone bond formation between a cationic peptide scaffold and a hydrophobic aldehyde tail. These peptide/protein non-covalent nanoparticles demonstrated comparable efficacy to leading methods while exhibiting reduced toxicity. To our knowledge, this represents the first supramolecular approach for direct Cas9 delivery utilizing a penetrating peptide carrier, streamlining the crucial step of cell membrane transport.
The findings presented here confirm that amphiphilic peptide vectors can effectively deliver Cas9 in a single incubation step, achieving high efficiency and low toxicity. This work paves the way for the exploration and development of novel synthetic systems for the transient delivery of endonucleases, enhancing cell membrane transport for therapeutic biomolecules.
