利用正向重复片段的GUS报告基因检测DNA双链断裂引起的基因重组研究

An Efficient Detecting System for Gene's Recombination Caused by DNA Double Strand Breaks Using Direct Reapeated Report Gene of GUS

本研究建立了一种方便快捷地检测DNA双链断裂引起的基因重组的方法。传统的检测DNA双链断裂的方法较为复杂、繁琐。因此,构建简单、快速的此类检测技术是非常必要的。本研究构建了利用正向重复片段的GUS基因载体以及引导RNA和噬菌体MS2外壳蛋白偶联限制性核酸内切酶的基因组编辑系统,在GUS基因内部DNA重复区切割DNA,导致双链断裂(double-strand breaks,DSBs),DSB经同源序列引导修复(homology-directed repair,HDR)途径,引起GUS基因重组,恢复其功能。此方法可以经过GUS染色后快速、直观地检测位点特异的DSB。通过GUS与其底物的反应,以达到半定量、直观快速检测DSB的目的。

In this study, a convenient and quick method for detecting gene recombination caused by DNA double strand breaks （doub-le-strand breaks,DSBs） was established. DNA DSBs can cause site-specific homology-directed repair（HDR）. However, the traditionalmethods of detecting DNA DSBs are complicated and cumbersome. Consequently, it is necessary to develope such a simple and fast detec-tion technique. This study was conducted by using the direct repeated GUS gene and guide RNA combined with phage MS2 coat protein cou-pled with restriction endonuclease genome editing system, in which gRNA is complementary with GUS repeat sequences. Based on produ-cing DSBs, functional GUS gene can be restored via HDR. By using the staining reaction of GUS with its substrate, the results can be a-chieved and quantitative detection of DSBs can be established. This method can be used quickly to detect site specific DSBs and intuitivelyobserve the results after GUS staining.