In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination

Nucleic Acids Res. 2005 Nov 23;33(20):e178. doi: 10.1093/nar/gni175.

Abstract

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • DNA / chemistry
  • DNA / metabolism
  • DNA Restriction Enzymes / chemistry
  • DNA Restriction Enzymes / genetics*
  • DNA Restriction Enzymes / metabolism
  • Gene Library
  • Genomics
  • Mutation
  • Peptide Library
  • Plasmids
  • Protein Engineering / methods*
  • Recombination, Genetic*
  • Saccharomyces cerevisiae / genetics

Substances

  • Peptide Library
  • DNA
  • DNA Restriction Enzymes
  • endodeoxyribonuclease CreI