Nanoblade-Based Delivery of Nucleic Acid Cargo: A Technique to Deliver Cas9-sgRNA Complex to Target Cells via Virus-Like Particles
Transcrição
Nanoblades are synthetic virus-like particles without the encapsulated viral genetic material, making them suitable carriers for foreign cargo into the host cells. These nanoblades are constructed using the retroviral core of the mouse leukemia virus surrounded by the lipid bilayer membrane derived from the eukaryotic producer cells used to assemble nanoblades in-vitro.
The core contains the Cas9 enzyme complexed with the sgRNA specific to the target genome sequence. Core proteins are surrounded by the envelope proteins that contain a transmembrane domain or TM that passes through the lipid bilayer. The TM attaches to an extracellular glycoproteinaceous surface domain or SU that protrudes from the bilayer coat.
To use nanoblades for gene editing, take a culture of adherent target cells and supplement it with the nanoblade solution. In the culture, the protruding SU of a nanoblade recognizes the corresponding receptor on the host cell surface and binds to it. This leads to modifications in the SU domain structure, facilitating its release.
These changes trigger the conformational changes in the TM domain, which then binds to the host membrane. This binding brings the nanoblade near the host cell, allowing the fusion of the viral bilayer membrane with the host membrane, and the viral core moves into the cytoplasm.
Once inside the cell, the core disintegrates, releasing the Cas9-sgRNA complex that can perform genome editing at its target site.
