title: Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.
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.
authors.affiliation: CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Arnould
authors.forename: Patrick
authors.initials: P
authors.lastname: Chames
authors.forename: Christophe
authors.initials: C
authors.lastname: Perez
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Lacroix
authors.forename: Aymeric
authors.initials: A
authors.lastname: Duclert
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Epinat
authors.forename: François
authors.initials: F
authors.lastname: Stricher
authors.forename: Anne-Sophie
authors.initials: AS
authors.lastname: Petit
authors.forename: Amélie
authors.initials: A
authors.lastname: Patin
authors.forename: Sophie
authors.initials: S
authors.lastname: Guillier
authors.forename: Sandra
authors.initials: S
authors.lastname: Rolland
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.forename: Jerónimo
authors.initials: J
authors.lastname: Bravo
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D016000:Cluster Analysis; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D011995:Recombination, Genetic; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI