Manipulation of 7-Finger Zinc Finger Nuclease Increases the Efficiency of Genome Editing in Human Cells

Genome editing tools have great potential for medicinal use. Among them, zinc finger nucleases (ZFNs) are smaller in size than transcriptional activator-like effector nucleases and CRISPR-Cas9. Therefore, ZFNs are easily packed into a viral vector with limited cargo space, including adeno-associated viral vectors. Furthermore, because ZFN patents expired in 2020, high patent royalties are not required for application. Although functional 6-finger ZFNs can be easily prepared by modular assembly, it has been extremely difficult to produce functional 7-finger ZFNs, which are expected to have higher target specificity than 6-finger ZFNs in some cases. Herein we describe the construction of 7-finger ZFNs and the improvement in genome editing efficiency, which is generally lower in 7-finger ZFNs than in 6-finger ZFNs. Modular assembly of 7-finger ZFNs was achieved using a specific mutation, and the original genome editing efficiency was increased by up to 19%. Furthermore, 7-finger ZFNs showed reduced off-target effects, exhibiting higher target specificity than the corresponding 6-finger ZFNs. Our study provides critical insights for safer and more specific genome editing.