Cationic Polymer-Mediated CRISPR/Cas9 Plasmid Delivery for Genome Editing

Area of Science:

• Biotechnology
• Molecular Biology
• Gene Editing

Background:

• CRISPR-Cas9 technology holds immense therapeutic potential but faces delivery challenges.
• Large plasmid sizes encoding Cas9 and sgRNA limit the efficiency of current delivery systems.
• Efficient delivery of genome editing tools is crucial for clinical translation.

Purpose of the Study:

• To evaluate the efficacy of a novel cationic polymer, polyethyleneimine-β-cyclodextrin (PC), for delivering large CRISPR-Cas9 plasmids.
• To demonstrate efficient genome editing using PC as a delivery vehicle.
• To establish a new strategy for overcoming plasmid delivery hurdles in genome editing.

Main Methods:

• Utilized polyethyleneimine-β-cyclodextrin (PC) as a cationic polymer carrier.
• Condensed and encapsulated large plasmids encoding Cas9 and single-guide RNA (sgRNA).
• Assessed genome editing efficiency at specific loci (hemoglobin subunit beta and RHBDF1) using Sanger sequencing.

Main Results:

• PC demonstrated efficient delivery of large plasmids encoding Cas9 and sgRNA.
• Achieved significant genome editing efficiencies at the hemoglobin subunit beta locus (19.1%) and RHBDF1 locus (7.0%).
• Sanger sequencing confirmed successful editing events at the targeted genomic sites.

Conclusions:

• Polyethyleneimine-β-cyclodextrin (PC) is an effective non-viral vector for delivering large CRISPR-Cas9 plasmids.
• This approach offers a promising strategy for advancing genome editing technologies towards clinical applications.
• Efficient delivery of CRISPR-Cas9 components is achievable with optimized polymer-based carriers.