Qi Yiping, Starker Colby G, Zhang Feng, Baltes Nicholas J, Voytas Daniel F
Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
Methods Mol Biol. 2014;1062:193-209. doi: 10.1007/978-1-62703-580-4_10.
Zinc finger nucleases (ZFNs) are proteins engineered to make site-specific double-strand breaks (DSBs) in a DNA sequence of interest. Imprecise repair of the ZFN-induced DSBs by the nonhomologous end-joining (NHEJ) pathway results in a spectrum of mutations, such as nucleotide substitutions, insertions, and deletions. Here we describe a method for targeted mutagenesis in Arabidopsis with ZFNs, which are engineered by context-dependent assembly (CoDA). This ZFN-induced mutagenesis method is an alternative to other currently available gene knockout or knockdown technologies and is useful for reverse genetic studies.
锌指核酸酶（ZFNs）是经过工程改造的蛋白质，可在感兴趣的DNA序列中产生位点特异性双链断裂（DSB）。非同源末端连接（NHEJ）途径对ZFN诱导的DSB进行不精确修复会导致一系列突变，如核苷酸替换、插入和缺失。本文我们描述了一种利用通过上下文依赖组装（CoDA）工程改造的ZFNs在拟南芥中进行靶向诱变的方法。这种ZFN诱导的诱变方法是其他现有基因敲除或敲低技术的替代方法，可用于反向遗传学研究。
