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

Authors
Journal
Nucleic Acids Research
0305-1048
Publisher
Oxford University Press
Publication Date
Volume
33
Issue
20
Identifiers
DOI: 10.1093/nar/gni175
Keywords
  • Methods Online
Disciplines
  • Engineering

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.

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