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

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...

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Autores principales: Fabien Delacôte, Christophe Perez, Valérie Guyot, Marianne Duhamel, Christelle Rochon, Nathalie Ollivier, Rachel Macmaster, George H Silva, Frédéric Pâques, Fayza Daboussi, Philippe Duchateau
Formato: article
Lenguaje:EN
Publicado: Public Library of Science (PLoS) 2013
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Acceso en línea:https://doaj.org/article/f3cd0e8d8ff24ecf88c47733d9ec4918
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Sumario: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.