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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Bibliographic Details
Published inPloS one Vol. 8; no. 1; p. e53217
Main Authors Delacôte, Fabien, Perez, Christophe, Guyot, Valérie, Duhamel, Marianne, Rochon, Christelle, Ollivier, Nathalie, Macmaster, Rachel, Silva, George H, Pâques, Frédéric, Daboussi, Fayza, Duchateau, Philippe
Format Journal Article
LanguageEnglish
Published United States Public Library of Science 24.01.2013
Public Library of Science (PLoS)
Subjects
Animals
Base Sequence
Biology
Cell cycle
CHO Cells
Cricetinae
Cricetulus
Deactivation
Deoxyribonucleic acid
DNA
DNA - genetics
DNA - metabolism
DNA damage
DNA End-Joining Repair
DNA Primers
Endonucleases - metabolism
Enzymes
Fibroblasts
Gene expression
Gene mutation
Genetic engineering
Genetic research
Genomes
HEK293 Cells
Homology
Humans
Hydrolases
Inactivation
Innovations
Life Sciences
Mutagenesis
Non-homologous end joining
Nuclease
Penicillin
Properties
Site-directed mutagenesis
Stem cells
Targeted mutagenesis
France
United States > US
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Summary: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.
Bibliography:Competing Interests: The authors have read the journal's policy and have the following conflicts: all co-authors are Cellectis employees. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: F. Delacôte CP RM GS PD F. Daboussi. Performed the experiments: F. Delacôte CP VG MD CR NO RM F. Daboussi. Analyzed the data: GS F. Daboussi PD FP. Contributed reagents/materials/analysis tools: RM MD. Wrote the paper: F. Delacôte CP PD FP.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0053217