Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects
View/ Open
Author
Certo, Michael T.
Mussolino, Claudio
Goodwin, Matthew J.
Cradick, Thomas J.
McCaffrey, Anton P.
Cathomen, Toni
Scharenberg, Andrew M.
Published Version
https://doi.org/10.1093/nar/gks179Metadata
Show full item recordCitation
Ramirez, Cherie L., Michael T. Certo, Claudio Mussolino, Mathew J. Goodwin, Thomas J. Cradick, Anton P. McCaffrey, Toni Cathomen, Andrew M. Scharenberg, and J. Keith Joung. 2012. Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects. Nucleic Acids Research 40(12): 5560-5568.Abstract
Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.Other Sources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384306/pdf/Terms of Use
This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAACitable link to this page
http://nrs.harvard.edu/urn-3:HUL.InstRepos:10445560
Collections
- HMS Scholarly Articles [17922]
Contact administrator regarding this item (to report mistakes or request changes)