Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast
Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast
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DOI：
10.1073/pnas.2036296100
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发表时间：
2003-12-09
影响因子：
11.1
通讯作者：
Resnick, MA
中科院分区：
文献类型：
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作者：
Storici, F;Durham, CL;Resnick, MA
在大多数生物体中，染色体双链断裂（DSB）的修复可以通过同源重组来完成。我们在此报告，外源寡核苷酸可以有效地靶向修复酵母染色体中诱导的单个 DSB。导致所需 DNA 变化的重组靶向效率可高达 20% 的细胞。 DSB 是在转化前由可调节的 I-Scel 核酸内切酶产生的，或者自发出现在由人 AN 序列组成的长反向重复序列的位点。该方法使用我们之前描述的 delitto perfetto 系统的特征来选择具有整合重组寡核苷酸的转化体。由互补整合重组寡核苷酸对介导的 DSB 修复对于靶向接近或远离 DSB 的同源序列以及在二倍体细胞中存在竞争性同源染色体的情况下是有效的。我们还证明 DSB 可以强烈刺激单链 DNA 的重组，而不会产生链偏向。这些发现扩展了当前的 DSB 修复模型。此外，我们建立了一个高通量系统，用于用寡核苷酸快速进行全基因组修饰。
The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms. We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells. The DSB was generated either by a regulatable I-Scel endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human AN sequences. The approach used features of our previously described delitto perfetto system for selecting transformants with integrative recombinant oligonucleotides. The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells. We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias. These findings expand current models of DSB repair. In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides.