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

Genome editing is commonly used in biomedical research. Among the genome editing tools, zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space. However, ZFNs also consist of left and right monomers, which both need to be expressed in the target cells. When each monomer is expressed separately, two expression cassettes are required, thus increasing the size of the DNA. This is a disadvantage for a viral vector with limited cargo space. We herein showed that T2A-coupled ZF-ND1 monomers were co-expressed from a single expression cassette and that the corresponding ZF-ND1s efficiently cleaved the target DNA sequences. Furthermore, the total amount of transfected plasmid DNA was reduced by half, and genome editing efficiency was equivalent to that of two separate ZF-ND1 monomers. This study provides a promising framework for the development of ZFN applications.