Engineering of Large Numbers of Highly Specific Homing Endonucleases that Induce Recombination on Novel DNA Targets

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• 作者：Sylvain Arnould ; Patrick Chames ; Christophe Perez ; Emmanuel Lacroix ; Aymeric Duclert ; Jean-Charles Epinat ; Franç ; ois Stricher ; Anne-Sophie Petit ; Amé ; lie Patin ; Sophie Guillier et al.
• 刊名：Journal of Molecular Biology
• 出版年：2006
• 出版时间：20 January 2006
• 年：2006
• 卷：355
• 期：3
• 页码：443-458
• 全文大小：932 K

文摘

The last decade has seen the emergence of a universal method for precise and efficient genome engineering. This method relies on the use of sequence-specific endonucleases such as homing endonucleases. The structures of several of these proteins are known, allowing for site-directed mutagenesis of residues essential for DNA binding. Here, we show that a semi-rational approach can be used to derive hundreds of novel proteins from I-CreI, a homing endonuclease from the LAGLIDADG family. These novel endonucleases display a wide range of cleavage patterns in yeast and mammalian cells that in most cases are highly specific and distinct from I-CreI. Second, rules for protein/DNA interaction can be inferred from statistical analysis. Third, novel endonucleases can be combined to create heterodimeric protein species, thereby greatly enhancing the number of potential targets. These results describe a straightforward approach for engineering novel endonucleases with tailored specificities, while preserving the activity and specificity of natural homing endonucleases, and thereby deliver new tools for genome engineering.