Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Nucleic Acids Res. 2012 Jul;40(12):5560-8. doi: 10.1093/nar/gks179. Epub 2012 Feb 28.

Abstract

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.

Publication types

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

MeSH terms

  • Cell Line
  • DNA Cleavage
  • DNA End-Joining Repair
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • HEK293 Cells
  • Humans
  • Mutagenesis
  • Protein Engineering / methods
  • Recombinational DNA Repair*
  • Zinc Fingers

Substances

  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • endodeoxyribonuclease FokI
  • Deoxyribonucleases, Type II Site-Specific