Precision genome engineering with programmable DNA-nicking enzymes

Genome Res. 2012 Jul;22(7):1327-33. doi: 10.1101/gr.138792.112. Epub 2012 Apr 20.

Abstract

Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs), which gives rise to randomly generated, unwanted small insertions or deletions (indels) at both on-target and off-target sites. Here, we present programmable DNA-nicking enzymes (nickases) that produce single-strand breaks (SSBs) or nicks, instead of DSBs, which are repaired by error-free homologous recombination (HR) rather than mutagenic NHEJ. Unlike their corresponding nucleases, zinc finger nickases allow site-specific genome modifications only at the on-target site, without the induction of unwanted indels. We propose that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • DNA Breaks, Double-Stranded
  • DNA Breaks, Single-Stranded
  • DNA End-Joining Repair
  • Deoxyribonuclease I / genetics
  • Deoxyribonuclease I / metabolism*
  • Deoxyribonucleases, Type II Site-Specific / genetics
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Gene Targeting / methods
  • Genetic Engineering / methods*
  • Genetic Vectors
  • Genome, Human*
  • HEK293 Cells
  • Humans
  • INDEL Mutation
  • Sensitivity and Specificity
  • Zinc Fingers*

Substances

  • endodeoxyribonuclease FokI
  • Deoxyribonuclease I
  • Deoxyribonucleases, Type II Site-Specific