Nuclear-targeted carbon quantum dot mediated CRISPR/Cas9 delivery for fluorescence visualization and efficient...

Area of Science:

• Biotechnology and Nanomedicine
• Gene Editing and Delivery Systems

Background:

• Efficient nuclear delivery of CRISPR/Cas9 systems is crucial for non-viral gene editing.
• Current methods face challenges in direct and effective delivery into the cell nucleus.

Purpose of the Study:

• To develop a nuclear-targeted gene delivery platform for CRISPR/Cas9.
• To enhance CRISPR/Cas9 delivery efficiency and genome editing outcomes.
• To evaluate the platform's safety and compare it with existing methods.

Main Methods:

• Fabrication of fluorescent carbon quantum dots (CQDs) passivated with polyethylenimine (PEI) and polyethylene glycol (PEG) to form CQDs-PEI-PEG (CQDs-PP).
• Complexation of CRISPR/Cas9 plasmid with CQDs-PP via electrostatic attraction.
• Delivery of the CRISPR/Cas9 nano-complex into HeLa cells and assessment of gene editing efficiency and cytotoxicity.

Main Results:

• CQDs-PP successfully formed nano-complexes with CRISPR/Cas9 plasmid.
• The nano-complexes bypassed lysosomes and entered the nucleus via passive diffusion, improving transfection.
• CQDs-PP demonstrated higher gene editing efficiency for the EFHD1 gene with comparable or lower cytotoxicity than Lipo2000 and CQDs-P.

Conclusions:

• The developed nuclear-targeted CQDs-PP platform is an effective carrier for CRISPR/Cas9 delivery.
• This platform offers improved gene editing efficiency and imaging-trackable capabilities.
• CQDs-PP presents a promising tool for in vitro genome editing applications.