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

Nat Biotechnol. 2007 Jul;25(7):778-85. doi: 10.1038/nbt1319. Epub 2007 Jul 1.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Biotechnology / methods*
  • Catalysis
  • Deoxyribonucleases, Type II Site-Specific / chemistry
  • Dimerization
  • Genome
  • Green Fluorescent Proteins / chemistry
  • Humans
  • K562 Cells
  • Models, Biological
  • Molecular Conformation
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Zinc Fingers*

Substances

  • Green Fluorescent Proteins
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific