Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds.
Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities. We demonstrate the modularity of the chimeric DmoCre meganuclease previously described, by successfully assembling mutants with locally altered specificities affecting both I-DmoI and I-CreI subdomains in order to create active meganucleases with altered specificities. Moreover these new engineered DmoCre variants appear highly specific and present a low toxicity level, similar to I-SceI, and can induce efficient homologous recombination events in mammalian cells. The DmoCre based meganucleases can therefore offer new possibilities for various genome engineering applications.
Grizot
Sylvestre
S
Cellectis SA, 102 Avenue Gaston Roussel, 93235 Romainville Cedex, France.
Epinat
Jean-Charles
JC
Thomas
Séverine
S
Duclert
Aymeric
A
Rolland
Sandra
S
Pâques
Frédéric
F
Duchateau
Philippe
P
D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D020650:Combinatorial Chemistry Techniques; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI; C081755:endodeoxyribonuclease I-Dmo I; D015253:Deoxyribonucleases, Type I Site-Specific