RNA editing using the flap endonuclease 1 guided by hairpin DNA probes. RNA editing using the flap endonuclease 1 guided by hairpin DNA probes

Here we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, called the HpSGN system, for both DNA and RNA editing without sequence limitation.To investigate the mRNA on/off target cleavage effective of the HpSGN system,we co-transformed HepG2 (liver hepatocellular cells) with plasmids encoding FEN1 with NES and two groups of hpDNAs targeting the mRNA of AFP (Alpha Fetoprotein) gene and CDK9 (Cyclin-dependent kinase 9) gene, respectively. RNA-seq was used to examine the on/off taget cleavage effective.We proved the HpSGN system can knock down specific mRNAs in human cells at a level of ~25%.At the same time,it indicated that a degree of off-targets occur in HpSGN-treated cells. Overall design: Examination the on/off target cleavage effect of the HpSGN system.

本研究对一种名为HpSGN系统的瓣状核酸内切酶1（flap endonuclease 1, FEN1）联合发夹DNA探针（hairpin DNA probe, hpDNA）体系进行了表征，该体系可实现无序列限制的DNA与RNA编辑。为探究HpSGN系统对mRNA的靶标与脱靶切割效率，我们将编码携带核输出信号（nuclear export signal, NES）的FEN1的质粒，与分别靶向甲胎蛋白（Alpha Fetoprotein, AFP）基因和细胞周期蛋白依赖性激酶9（Cyclin-dependent kinase 9, CDK9）基因mRNA的两组hpDNA，共转染至HepG2（肝实质细胞）细胞中。采用RNA-seq技术检测靶标与脱靶切割效率。本研究证实，HpSGN系统可在人类细胞中敲低约25%的特异性mRNA。同时，研究结果显示经HpSGN处理的细胞中存在一定程度的脱靶效应。实验整体设计：探究HpSGN系统对mRNA的靶标与脱靶切割效应。