Design and High-Throughput Generation of Artificial Small RNA Constructs for Plants
Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of 21-nucleotide artificial small RNAs (sRNAs) designed to selectively silence transcripts in plants with high efficacy and specificity. Despite their extensive use during the last decade, methods for designing and generating artificial sRNA constructs have not been optimized for time- and cost-effectiveness and high-throughput applicability since recently. In this chapter, I detail the protocols for both the rationale design and high-throughput generation of plant artificial sRNA constructs using the P-SAMS (“Plant Small RNA Maker Suite”) web tool and a new generation of BsaI/ccdB (B/c) vectors optimized for one-step cloning of artificial sRNA inserts. These protocols allow for the efficient generation of large number of amiRNA and syn-tasiRNA constructs for potent, selective, and specific gene silencing in plants.
Key wordsArtificial small RNA amiRNA syn-tasiRNA RNA silencing P-SAMS
This work was supported by grants BIO2014-54269-R and BIO2017-83184-R from the Ministerio de Economía, Industria y Competitividad (Spain). Alberto Carbonell was the recipient of an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement No. 655841, and was selected in the Ramón y Cajal programme (RYC-2017-21648) from Ministerio de Ciencia, Innovación y Universidades (Spain).
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