Title | Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. |
Publication Type | Journal Article |
Year of Publication | 2006 |
Authors | Arnould S, Chames P, Perez C, Lacroix E, Duclert A, Epinat J-C, Stricher F, Petit A-S, Patin A, Guillier S, Rolland S, Prieto J, Blanco FJ, Bravo J, Montoya G, Serrano L, Duchateau P, Pâques F |
Journal | J Mol Biol |
Volume | 355 |
Issue | 3 |
Pagination | 443-58 |
Date Published | 2006 Jan 20 |
ISSN | 0022-2836 |
Keywords | Amino Acid Sequence, Animals, Base Sequence, CHO Cells, Cluster Analysis, Cricetinae, Cricetulus, Dimerization, DNA, DNA Restriction Enzymes, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Engineering, Recombination, Genetic, Yeasts |
Abstract | 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. |
DOI | 10.1016/j.jmb.2005.10.065 |
Alternate Journal | J. Mol. Biol. |
PubMed ID | 16310802 |