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 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.
Cellectis SA, Paris, France.
Fabien
F
Delacôte
Christophe
C
Perez
Valérie
V
Guyot
Marianne
M
Duhamel
Christelle
C
Rochon
Nathalie
N
Ollivier
Rachel
R
Macmaster
George H
GH
Silva
Frédéric
F
Pâques
Fayza
F
Daboussi
Philippe
P
Duchateau
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D059766:DNA End-Joining Repair; D017931:DNA Primers; D004720:Endonucleases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis
D017931:DNA Primers; D004247:DNA; D004720:Endonucleases