Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects.
Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects.
复制标题
DOI：
10.1093/nar/gks179
复制
发表时间：
2012-07
影响因子：
14.9
通讯作者：
Joung JK
中科院分区：
文献类型：
--
作者：
Ramirez CL;Certo MT;Mussolino C;Goodwin MJ;Cradick TJ;McCaffrey AP;Cathomen T;Scharenberg AM;Joung JK
工程锌指核酸酶（ZFNs）在特定的识别序列上诱导DNA双链断裂，并通过同源定向修复（HDR）与双链和/或单链供体DNA模板在切割位点或附近有效地引入所需的插入、缺失或取代。然而，易出错的非同源末端连接（NHEJ）介导的修复引起的诱变事件在核酸酶切割位点以相同或更高的频率引入。此外，nhej介导的脱靶核酸酶切割位点的修复也可能导致意想不到的突变。在这里，我们描述了一种简单而通用的方法，通过失活ZFN二聚体中一个单体的催化活性，将工程ZFN转化为锌指缺口酶（ZFNickases）。zfnickase在体外表现出强大的链特异性切口活性。此外，我们证明zfnickase可以在人类细胞的缺口位点刺激HDR，尽管其频率低于其来源的ZFNs。最后，我们发现zfnickase在其目标切口位点诱导的诱变NHEJ水平大大降低。因此，zfnickase为诱导hdr介导的基因修饰提供了一种有希望的方法，同时减少了易出错的NHEJ引起的不必要的突变。
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.
登录
查看更多内容
DOI：
10.1016/s0027-5107(98)00051-7
发表时间：
1998-05-25
影响因子：
2.3
作者：
Holmquist, GP
通讯作者：
Holmquist, GP
影响因子：
12.4
作者：
Alwin, S;Gere, MB;Cathomen, T
通讯作者：
Cathomen, T
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
12.4
作者：
Cornu, Tatjana I.;Thibodeau-Beganny, Stacey;Cathomen, Toni
通讯作者：
Cathomen, Toni
影响因子：
46.9
作者：
Gabriel, Richard;Lombardo, Angelo;von Kalle, Christof
通讯作者：
von Kalle, Christof