pssRNAit : A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment

作者：Firoz Ahmed, Muthappa Senthil‐Kumar, Xinbin Dai, Ramu S. Vemanna, Seonghee Lee, Kirankumar S. Mysore, Patrick X. Zhao · 发表于：PLANT PHYSIOLOGY · 年份：2020 · DOI：10.1104/pp.20.00293 · 被引用次数：74 · 研究领域：RNA Interference and Gene Delivery、Plant Virus Research Studies、Chromosomal and Genetic Variations

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, 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 pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana. 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 pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit. The pssRNAit 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 hig...