Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways
Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways
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通过操纵 DNA 修复途径增加拟南芥中定点诱变和基因靶向的频率
DOI：
10.1101/gr.145557.112
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发表时间：
2013-03-01
期刊：
影响因子：
7
通讯作者：
Voytas, Daniel F.
中科院分区：
文献类型：
--
作者：
Qi, Yiping;Zhang, Yong;Voytas, Daniel F.
用于工程化序列特异性核酸酶（包括锌指核酸酶（ZFN）和TAL效应物核酸酶（TALEN））的改进方法已经使得精确修饰植物基因组成为可能。然而，基因组修饰的成功在很大程度上取决于工程化核酸酶的内在活性。在这项研究中，我们试图提高ZFN介导的定向诱变和基因打靶（GT）在拟南芥中通过操纵DNA修复途径。使用在内源性ADH 1位点产生双链断裂（DSB）的ZFN，我们分析了在不存在DNA修复蛋白（如KU70和LIG4（均参与经典的非同源末端连接，NHEJ）和SMC6B（参与基于姐妹染色单体的同源重组，HR））的情况下的修复结果。我们在ku70突变体中实现了基于HR的GT的5倍至16倍增强，在lig4突变体中实现了GT的3倍至4倍增强。虽然在ku70或lig4中NHEJ诱变频率没有显著改变，但DNA修复转移到微同源性依赖的替代NHEJ。结果，ku70和lig4中的突变主要是大缺失，这有助于通过PCR容易地筛选突变。有趣的是，在smc6b突变体中，ADH 1位点的NHEJ诱变和GT分别增强了6倍至8倍和3倍至4倍。使用靶向另外两个拟南芥基因（即TT4和MPK 8）的ZFN进一步证实了通过丢失SMC6B引起的NHEJ介导的诱变的增加。考虑到DNA修复途径的组分在物种间高度保守，DNA修复基因中的突变可能提供了利用修复途径实现所需靶向基因组修饰的通用策略。
Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications.