| The Making of Novel Artificial Meganucleases Derived from Homing Endonucleases to Induce Homologous Recombination in Living Cells |
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Presentation: P03006 Session: Robotic Screening & Automation Technologies - Poster SessionJean-Charles Epinat, Patrick Chames, Sylvain Arnould, Emmanuel Lacroix, Aymeric Duclert, Philippe Duchateau and Frédéric Paques, CELLECTIS, S.A. Presenting Author: Jean-Charles Epinat, CELLECTIS - France Meganucleases are sequence-specific endonucleases with long (14-40 bp) target sequences. They produce DNA double-strand breaks thereby enhancing gene targeting efficiency around the cleavage site by several orders of magnitude in both mammalian cells and plants. Using a semi-rational approach and a high-throughput assay that directly monitors Meganuclease-induced recombination in living cells, we derived hundreds of Meganucleases with new specificities from the I-CreI natural Homing Endonuclease. Clear cut switches of specificity could be observed: many new proteins displayed high levels of cleavage on new targets, but no detectable activity on the wild type cleavage sites. Cleavage profiles of the new proteins on a large number of DNA sequences also demonstrated that most novel proteins had kept a very narrow specificity. Our approach shows the potential to evolve the specificity of meganucleases, thereby delivering new endonucleases with the high levels of activity and specificity required to perform rational genome engineering. |