Efficient Genome Editing Using the T2A-Coupled Co-Expression of Two ZFN Monomers

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biotechnology

Background:

• Genome editing is a crucial tool in biomedical research.
• Zinc finger nucleases (ZFNs) are smaller than other genome editing tools like TALENs and CRISPR-Cas9, making them suitable for viral vectors.
• A limitation of ZFNs is the need for separate expression of left and right monomers, requiring two DNA cassettes, which increases vector size.

Purpose of the Study:

• To develop a method for co-expressing ZFN monomers from a single expression cassette.
• To overcome the size limitation of viral vectors for ZFN delivery.
• To maintain or improve genome editing efficiency with the new co-expression system.

Main Methods:

• Engineered T2A-coupled ZF-ND1 monomers for co-expression from a single DNA cassette.
• Transfection of the single expression cassette into target cells.
• Assessed DNA cleavage efficiency and compared genome editing outcomes with a two-cassette system.

Main Results:

• Successfully demonstrated co-expression of ZF-ND1 monomers using a single T2A-coupled expression cassette.
• The co-expressed ZF-ND1s efficiently cleaved target DNA sequences.
• Reduced the total transfected plasmid DNA amount by half while achieving equivalent genome editing efficiency compared to two separate monomers.

Conclusions:

• Co-expression of ZFN monomers via a single T2A-coupled cassette is feasible and efficient.
• This strategy significantly reduces DNA payload size, benefiting viral vector applications.
• Provides a promising framework for advancing ZFN-based genome editing technologies.