Quadruple pegRNA enables programmable and efficient large genomic insertion

Area of Science:

• Gene editing and therapy
• Molecular biology
• Genomics

Background:

• Precise gene insertion is crucial for treating genetic disorders.
• Current prime editing methods struggle with large DNA payloads (>300 bp).

Purpose of the Study:

• To develop an efficient and programmable method for inserting large DNA fragments using prime editing.
• To overcome the limitations of existing gene insertion technologies.

Main Methods:

• Designed a quadruple pegRNA (paired guide RNA) strategy (QuadPE).
• Screened pegRNA configurations (genome-targeting and donor-targeting) for optimal efficiency.
• Tested QuadPE for inserting DNA fragments from 1.6 kb to 26 kb at multiple genomic loci.

Main Results:

• QuadPE achieved stable integration efficiencies of ~40% for large DNA fragments (up to 26 kb).
• Demonstrated significantly higher efficiency compared to recombinase-mediated (PASSIGE, PASTE) and transposase-mediated (CAST) systems.
• Showed effectiveness in both dividing and non-dividing primary cells (e.g., T cells, neurons) with minimal off-target activity.

Conclusions:

• QuadPE is a powerful and precise platform for large-fragment gene insertion.
• This method does not require double-stranded breaks or recombinases, offering a safer alternative.
• QuadPE advances gene therapy development for a wider range of genetic disorders.