An improved zinc-finger nuclease architecture for highly specific genome editing
An improved zinc-finger nuclease architecture for highly specific genome editing
复制标题
DOI：
10.1038/nbt1319
复制
发表时间：
2007-07-01
影响因子：
46.9
通讯作者：
Rebar, Edward J.
中科院分区：
文献类型：
--
作者：
Miller, Jeffrey C.;Holmes, Michael C.;Rebar, Edward J.
由锌指核酸酶（ZFN）驱动的基因组编辑通过将重组双链断裂引入靶基因中而产生高基因修饰效率（> 10%）。使用两个定制设计的ZFN诱导切割事件，所述ZFN在结合DNA时异源二聚化以形成催化活性的核酸酶复合物。然而，使用目前的ZFN结构，也可以形成可切割的同二聚体，其可以通过脱靶切割限制安全性或功效。在这里，我们开发了一个改进的ZFN架构，消除了这个问题。使用基于结构的设计，我们设计了两种变体ZFN，它们仅在作为异二聚体配对时才能有效地切割DNA。这些ZFN与亲本结构一样有效地修饰天然内源基因座，但同源二聚体功能降低> 40倍，基因组范围的切割水平低得多。这种结构提供了用于改善ZFN作为基因修饰试剂的特异性的一般手段。
Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (> 10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off- target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a > 40-fold reduction in homodimer function and much lower levels of genome- wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.