Published online 23 November 2005
Methods Online |
In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination
CELLECTIS S.A. 102 route de Noisy 93235 Romainville, France
*To whom correspondence should be addressed. Tel: +33 1 41 83 99 00; Fax: +33 1 41 83 99 03; Email: paques{at}cellectis.com
Received September 5, 2005. Revised October 21, 2005. Accepted October 21, 2005.
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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