Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets.
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.
Arnould
Sylvain
S
CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France.
Chames
Patrick
P
Perez
Christophe
C
Lacroix
Emmanuel
E
Duclert
Aymeric
A
Epinat
Jean-Charles
JC
Stricher
François
F
Petit
Anne-Sophie
AS
Patin
Amélie
A
Guillier
Sophie
S
Rolland
Sandra
S
Prieto
Jesús
J
Blanco
Francisco J
FJ
Bravo
Jerónimo
J
Montoya
Guillermo
G
Serrano
Luis
L
Duchateau
Philippe
P
Pâques
Frédéric
F
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
D004247:DNA; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI