High frequency targeted mutagenesis using engineered endonucleases and DNA-end processing enzymes

PLoS One. 2013;8(1):e53217. doi: 10.1371/journal.pone.0053217. Epub 2013 Jan 24.

Abstract

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

MeSH terms

  • Animals
  • Base Sequence
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • DNA / genetics
  • DNA / metabolism*
  • DNA End-Joining Repair*
  • DNA Primers
  • Endonucleases / metabolism*
  • HEK293 Cells
  • Humans
  • Mutagenesis*

Substances

  • DNA Primers
  • DNA
  • Endonucleases

Grants and funding

The authors have no support or funding to report.