Efficient Intracellular Delivery of CRISPR-Cas9 Ribonucleoproteins Using Dendrimer Nanoparticles for Robust Genomic...

Area of Science:

• Biotechnology
• Gene Editing
• Nanomedicine

Background:

• CRISPR-Cas9 gene editing is powerful but hindered by inefficient intracellular delivery of large ribonucleoprotein complexes.
• Current delivery methods suffer from toxicity, immunogenicity, poor scalability, and lack of cell specificity.

Purpose of the Study:

• To develop a novel, non-toxic dendrimer conjugate for efficient CRISPR-Cas9 delivery.
• To overcome the limitations of existing gene editing delivery vectors.

Main Methods:

• Covalent conjugation of Streptococcus pyogenes Cas9-2NLS (Cas9-nuclear localization sequence) to a hydroxyl PAMAM dendrimer using a glutathione-sensitive disulfide linker and inverse Diels-Alder click chemistry (IEDDA).
• Complexation of single guide RNA (sgRNA) to create a Cas9-dendrimer conjugate nano-construct (D-Cas9).
• In vitro assessment of D-Cas9-RNP efficacy in HEK293 and ARPE-19 cells.

Main Results:

• Achieved robust genomic deletion of GFP (~100%) in HEK293 cells and VEGF (20%) in ARPE-19 cells.
• Demonstrated rapid cellular uptake of D-Cas9-RNP constructs, comparable to small molecules.
• The neutral dendrimer construct exhibited enhanced stability and retained Cas9 payload functionality.

Conclusions:

• The developed dendrimer conjugate represents a promising, non-toxic, and efficient platform for CRISPR-Cas9 delivery.
• This approach may advance neutral dendrimer-enabled, cell-specific genome editing both in vitro and in vivo.
• The conjugation strategy offers improved stability and payload delivery for gene editing applications.