High frequency targeted mutagenesis using engineered endonucleases and DNA-End processing enzymes

Fabien Delacôte (1) , Christophe Perez (1) , Valérie Guyot (1) , Marianne Duhamel (2) , Christelle Rochon (2) , Nathalie Ollivier (2) , Rachel Macmaster (1) , George H. Silva (1) , Frédéric Pâques (1) , Fayza Daboussi (1) , Philippe Duchateau (1)

1 Cellectis SA
2 Genopole

Targeting DNA double-strand breaks is a powerful strategy for gene inactivation applications. Without the use of a repair plasmid, targeted mutagenesis can be achieved through Non-Homologous End joining (NHEJ) pathways. However, many of the DNA breaks produced by engineered nucleases may be subject to precise re-ligation without loss of genetic information and thus are likely to be unproductive. In this study, we combined engineered endonucleases and DNA-end processing enzymes to increase the efficiency of targeted mutagenesis, providing a robust and efficient method to (i) greatly improve targeted mutagenesis frequency up to 30-fold, and; (ii) control the nature of mutagenic events using meganucleases in conjunction with DNA-end processing enzymes in human primary cells.

Domaines: Sciences du Vivant [q-bio]

Fabien Delacôte, Christophe Perez, Valérie Guyot, Marianne Duhamel, Christelle Rochon, et al.. High frequency targeted mutagenesis using engineered endonucleases and DNA-End processing enzymes. PLoS ONE, 2013, 8 (1), 8 p. ⟨10.1371/journal.pone.0053217⟩. ⟨hal-01606192⟩