Department of Biochemistry, College of Science, University of Jeddah, Jeddah 21589, Saudi Arabia.
University of Jeddah Center for Scientific and Medical Research, University of Jeddah, Jeddah 21589, Saudi Arabia.
Plant Physiol. 2020 Sep;184(1):65-81. doi: 10.1104/pp.20.00293. Epub 2020 Jul 10.
We report an advanced web server, the plant-specific small noncoding RNA interference tool , which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of by designing and expressing the VIGS constructs to silence () or a ribosomal protein-encoding gene, (), in We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by , targeting different homologous regions of the gene. Our analyses confirmed the high accuracy of siRNA constructs designed using The server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.
我们报告了一个先进的植物特异性小非编码 RNA 干扰工具的网络服务器，该工具可用于设计一组小干扰 RNA（siRNA），以在植物中实现高效、特异和无毒的基因沉默。在开发这个工具时，我们整合了植物的转录数据集、几个基因沉默的规则以及 RNA 干扰（RNAi）途径的生物机制的一系列计算模型。设计的 siRNA 库可用于构建长双链 RNA，并通过病毒诱导的基因沉默（VIGS）或合成反式作用 siRNA 载体进行基因沉默表达。我们通过设计和表达 VIGS 构建体来沉默拟南芥中的（）或核糖体蛋白编码基因（），展示了 的性能。我们使用逆转录定量 PCR 分析了预测的目的靶基因和脱靶基因的表达水平。我们进一步进行了基于 RNA-seq 的转录组分析，以评估针对不同同源区域的基因设计的片段通过 引发的全基因组脱靶基因沉默。我们的分析证实了使用 设计的 siRNA 构建体具有很高的准确性。该服务器可免费在 https://plantgrn.noble.org/pssRNAit/ 获得，支持针对 >160 种植物物种进行高通量功能基因组学和性状改良的高效和特异的 RNAi、VIGS 或合成反式作用 siRNA 构建体的设计。
