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An improved zinc-finger nuclease architecture for highly specific genome editing

来自 Springer

阅读量：

506

作者：

JC Miller，MC Holmes，J Wang，DY Guschin，YL Lee，I Rupniewski，CM Beausejour，AJ Waite，NS Wang，KA Kim

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摘要：

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

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关键词：

Humans K562 Cells Deoxyribonucleases, Type II Site-Specific Green Fluorescent Proteins Base Sequence Molecular Conformation Zinc Fingers Protein Structure, Tertiary Dimerization Binding Sites

DOI：

10.1038/nbt1319

被引量：

1772

年份：

2007

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来源期刊

Nature Biotechnology

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2013
被引量：212

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