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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification
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Triplex-forming oligonucleotide-orthophenanthroline conjugates for efficient targeted genome modification

机译：三链体形成寡核苷酸-邻菲咯啉偶联物可有效靶向基因组修饰

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摘要

The inefficiency of gene modification by homologous recombination can be overcome by the introduction of a double-strand break (DSB) in the target. Engineering the endonucleases needed, however, remains a challenging task that limits widespread application of nuclease-driven gene modification. We report here that conjugates of orthophenanthroline (OP), a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences, are synthetic nucleases efficient at stimulating targeted genome modification. We show that in cultured cells, OP-TFO conjugates induce targeted DSBs. An OP-TFO with a unique target was highly efficient, and mutations at the target site were found in ≈10% of treated cells, including small deletions most likely introduced during DSB repair by nonhomologous end joining. Importantly, we found that when homologous donor DNA was cotransfected, targeted gene modification took place in >1.5% of treated cells. Because triplex-forming sequences are frequent in human and mouse genes, OP-TFO conjugates therefore constitute an important class of site-specific nucleases for targeted gene modification. Harnessing DNA-damaging molecules to predetermined genomic sites, as achieved here, should also provide inroads into mechanisms of DNA repair and cancer.
机译：通过在靶标中引入双链断裂（DSB），可以克服通过同源重组进行基因修饰的效率低下的问题。然而，工程化所需的核酸内切酶仍然是一项艰巨的任务，其限制了核酸酶驱动的基因修饰的广泛应用。我们在这里报告说，邻菲咯啉（OP），DNA切割分子和三链形成寡核苷酸（TFOs）的结合物，已知能结合特定的DNA序列，是有效刺激目标基因组修饰的合成核酸酶。我们显示，在培养的细胞中，OP-TFO共轭物可诱导靶向的DSB。具有独特靶标的OP-TFO是高效的，在约10％的处理细胞中发现了靶位点的突变，包括在非同源末端连接DSB修复过程中极有可能引入的小缺失。重要的是，我们发现将同源供体DNA共转染时，> 1.5％的已处理细胞发生了靶向基因修饰。由于三重形成序列在人和小鼠基因中很常见，因此OP-TFO缀合物构成了一类重要的位点特异性核酸酶，用于靶向基因修饰。如此处所实现的，将破坏DNA的分子利用到预定的基因组位点上，也应有助于侵入DNA修复和癌症的机制。

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