title: Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects.
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.
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA.
authors.forename: Cherie L
authors.initials: CL
authors.lastname: Ramirez
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.forename: Mathew J
authors.initials: MJ
authors.lastname: Goodwin
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Cradick
authors.forename: Anton P
authors.initials: AP
authors.lastname: McCaffrey
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D002460:Cell Line; D053837:DNA Cleavage; D059766:DNA End-Joining Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods; D059767:Recombinational DNA Repair; D016335:Zinc Fingers
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific