Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 210602, China.
Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Sci Rep. 2017 Mar 22;7(1):301. doi: 10.1038/s41598-017-00374-7.
It has been reported that double-stranded break (DSB)-induced small RNAs (diRNAs) are generated via the RNA-directed DNA methylation pathway and function in DSB repair in Arabidposis. However, important questions remain regarding the biogenesis and function of diRNAs. Here, we used CRISPR/Cas9- or TALEN-triggered DSBs to characterize diRNAs in Arabidopsis and rice. We found that 21-nt diRNAs were generated from a 35S promoter::GU-US reporter transgene targeted by CRISPR/Cas9. Unexpectedly, Pol II transcription of the transgene was required for efficient diRNA production and the level of diRNA accumulation correlated with the expression level of the transgene. diRNAs were not detected from CRISPR/Cas9- or TALEN-induced DSBs within the examined endogenous genes in Arabidopsis or rice. We also found that DCL4 and RDR6 that are known to be involved in posttranscriptional gene silencing were required to generate diRNAs. Our results suggest that DSBs are necessary but not sufficient for efficient diRNA generation and a high level of diRNAs is not necessary for DSB repair.
据报道,双链断裂(DSB)诱导的小 RNA(diRNAs)是通过 RNA 指导的 DNA 甲基化途径产生的,并且在拟南芥的 DSB 修复中发挥作用。然而,关于 diRNAs 的生物发生和功能仍存在一些重要问题。在这里,我们使用 CRISPR/Cas9 或 TALEN 触发的 DSB 来表征拟南芥和水稻中的 diRNAs。我们发现,21-nt diRNAs 是从 35S 启动子::GU-US 报告基因转基因中产生的,该转基因靶向 CRISPR/Cas9。出乎意料的是,转基因的 Pol II 转录对于高效的 diRNA 产生是必需的,并且 diRNA 的积累水平与转基因的表达水平相关。在拟南芥或水稻中,未从 CRISPR/Cas9 或 TALEN 诱导的 DSB 中检测到内源性基因内的 diRNAs。我们还发现,已知参与转录后基因沉默的 DCL4 和 RDR6 对于产生 diRNAs 是必需的。我们的结果表明,DSB 对于高效 diRNA 的产生是必要的,但不是充分的,并且高水平的 diRNAs 对于 DSB 修复不是必需的。