Engineering of Zinc Finger Nucleases Through Structural Modeling Improves Genome Editing Efficiency in Cells

Area of Science:

• Biomedical Research
• Molecular Biology
• Gene Editing Technologies

Background:

• Zinc finger nucleases (ZFNs) are crucial for genome editing, offering advantages in size over TALENs and CRISPR-Cas9.
• ZFNs' small size facilitates packaging into viral vectors like AAV for in vivo and clinical applications.
• Challenges exist in constructing functional ZFNs and enhancing their genome editing efficiency.

Purpose of the Study:

• To describe an efficient method for constructing functional ZFNs.
• To improve the genome editing efficiency of ZFNs.
• To demonstrate the utility of structural modeling tools in ZFN engineering.

Main Methods:

• Assembly of plasmids encoding six-finger ZFNs using public zinc-finger resources.
• Utilized AlphaFold, Coot, and Rosetta for engineering ZFNs.
• Tested ten ZFN constructs for functionality and editing efficiency.

Main Results:

• Successfully constructed two functional ZFNs out of ten tested.
• Engineering ZFNs with AlphaFold, Coot, or Rosetta enhanced genome editing efficiency by 5%.
• Structural modeling-based engineering proved effective for improving ZFN performance.

Conclusions:

• Efficient construction and engineering of ZFNs are achievable using structural modeling tools.
• The developed methods enhance ZFNs' potential for translational research and clinical applications.
• This work validates the use of AlphaFold, Coot, and Rosetta for optimizing genome editing tools.