Highly active zinc-finger nucleases by extended modular assembly.

Area of Science:

• Molecular Biology
• Genome Engineering
• Biotechnology

Background:

• Zinc-finger nucleases (ZFNs) are crucial for genome engineering but their widespread adoption is limited by the lack of robust, noncommercial methods.
• The modular assembly (MA) method allows rapid ZFN creation but often results in inactive nucleases, particularly with three- and four-finger arrays, with success rates below 25%.

Purpose of the Study:

• To systematically investigate the impact of array length on ZFN activity and efficiency.
• To identify optimal ZFN designs for enhanced genome targeting and success rates.

Main Methods:

• Systematic study of ZFN array lengths, focusing on three- to six-finger combinations.
• Development and application of a novel drop-out linker scheme for rapid assessment of ZFN combinations.
• Analysis of 268 array variants, correlating activity with an ab initio B-score cutoff.

Main Results:

• Six-finger MA ZFN arrays successfully produced mutations at 71% (15 of 21) of targeted loci in human and mouse cells.
• Shorter arrays demonstrated improved activity in specific cases.
• Half of MA ZFNs exceeding an ab initio B-score cutoff of 15 were active, irrespective of array composition.

Conclusions:

• Optimizing ZFN array length, particularly using longer six-finger arrays, significantly enhances the success rate of genome engineering.
• The MA method, when appropriately applied with optimized array lengths, offers a highly effective approach for targeting diverse DNA sequences.
• This research provides a pathway to more robust and successful genome engineering applications using ZFN technology.