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pssRNAit-a web server for designing effective and specific plant siRNAs with genome-wide off-target assessment

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Title pssRNAit-a web server for designing effective and specific plant siRNAs with genome-wide off-target assessment
 
Creator Ahmed, Firoz
Senthil-Kumar, Muthappa
Dai, Xinbin
Ramu, Vemanna S.
Lee, Seonghee
Mysore, Kirankumar S
Zhao, Patrick Xuechun
 
Subject pssRNAit
web server
plant siRNAs
 
Description Accepted date: June 30, 2020
We report an advanced web server named pssRNAit (plant specific small non-coding RNAi tool) that can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and non-toxic 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-stranded RNA (long-dsRNA) and expressed through virus-induced gene silencing (VIGS) or synthetic trans-acting siRNA (syn-tasiRNA) 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 RT-qPCR. We further conducted an RNA-seq-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 syn-tasiRNA constructs for high-throughput functional genomics and trait improvement in more than 160 plant species.
National Science Foundation [DBI-0960897 and DBI-1458597 to PXZ]; Oklahoma
Center for the Advancement of Science and Technology [PSB11-004 to PXZ]; Noble Research Institute. Funding for open access charge: National Science Foundation [DBI-0960897 and DBI 1458597 to PXZ]; Noble Research Institute.
 
Date 2020-07-13T09:55:38Z
2020-07-13T09:55:38Z
2020
 
Type Article
 
Identifier Plant Physiology, 184(1): 65-81
1532-2548
https://doi.org/10.1104/pp.20.00293
http://www.plantphysiol.org/content/early/2020/07/10/pp.20.00293
http://223.31.159.10:8080/jspui/handle/123456789/1073
 
Language en_US
 
Format application/pdf
 
Publisher American Society of Plant Biologists