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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| Main Authors: | , , , , , , , , , , |
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| Format: | article |
| Language: | EN |
| Published: |
Public Library of Science (PLoS)
2013
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| Subjects: | |
| Online Access: | https://doaj.org/article/f3cd0e8d8ff24ecf88c47733d9ec4918 |
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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. |
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