![]() However, data on the experimental validation of several different constructs used for silencing in plants are more limited. Due to the absence of the interferon response in plants, the use of long double-stranded RNA as a trigger of RNA is possible. This also influenced the development of experimental techniques and software tools. Therefore, the intention in animal and human research is to use short double-stranded oligonucleotides for triggering the silencing. In mammals, long double-stranded RNA molecules are known to induce strong interferon reactions and cell death ( Isaacs and Lindenmann, 1957). Also, software from commercial distributors is available. Several publicly available software tools, mainly intended for their use in mammals, are described for the design of dsRNA silencing inducers ( Naito and Ui-Tei, 2012 Schmidt et al., 2013). A vast amount of experimental data has been accumulated, which allows design and validation of RNAi prediction models ( McGinnis, 2010). RNAi has become widely used as a tool to modulate gene expression. Once the guide siRNA chain of the silencing complex is paired to the target mRNA molecule, the latter will be hydrolyzed, and thus, the expression of the corresponding gene will be knocked down ( Hammond et al., 2000 Bernstein et al., 2001). This interaction is probably influenced-besides other factors-by the physical accessibility and presence of unpaired nucleotides at the RNA target site ( Ameres et al., 2007). The mRNA targets are then found by nucleotide pairing between the antisense (guide) strand of the AGO-incorporated siRNA on the one side and the target mRNA on the other. A sequence asymmetry-sensing mechanism is selecting one strand to be incorporated into an ARGONAUT-containing complex named RISC (RNA-induced silencing complex) ( Khvorova et al., 2003 Schwarz et al., 2003 Amarzguioui and Prydz, 2004 Reynolds et al., 2004 Ui-Tei et al., 2004 Frank et al., 2010 Walton et al., 2010 Noland et al., 2011). They are generated by a DICER-mediated cleavage of double-stranded RNA (dsRNA) into 19–25 base pair (bp) long double-stranded oligonucleotides with 2-nt 3’overhangs. These small RNAs, usually 21 nucleotides (nt) in length, mediate the sequence specificity and represent the active principle of successful gene silencing ( Zamore et al., 2000 Elbashir et al., 2001). Small interfering RNAs (siRNAs) are the hallmark of posttranscriptional gene silencing (PTGS). PTGS is an essential part of plant immune response to viruses ( Muhammad et al., 2019) and required for genomic stability by silencing of retroelements ( Almeida and Allshire, 2005). RNAi can be used for stable as well as for transient transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS) ( Unniyampurath et al., 2016). As this application is based on sequence similarity of the silencing trigger to the respective targets, it allows silencing of several gene family members with a unique construct. Furthermore, the RNAi-mediated silencing is quantitative (knockdown) and can be directed to tissue or developmental specificity by the utilization of the respective promoter. RNAi is able to target any transcript regardless of ploidy, and it is not hampered by chromatin structure modifications ( Uusi-Makela et al., 2018). Although the novel technique of CRISPR/Cas9-directed site-specific mutagenesis is attracting wide scientific interest, RNAi still offers advantages. High-throughput gene silencing technologies were applied in several organisms to study gene function. si-Fi is an open-source (CC BY-SA license) desktop software that works in Microsoft Windows environment and can use custom sequence databases in standard FASTA format. It offers efficiency prediction of RNAi sequences and off-target search, required for the practical application of RNAi. Here, we present si-Fi, a software tool for design optimization of RNAi constructs necessary for specific target gene knock-down. RNAi has become an important research tool for studying gene function by strong and selective suppression of target genes. PTGS also mediates genomic stability by silencing of retroelements. PTGS is an ubiquitous basic biological phenomenon involved in the regulation of transcript abundance and plants’ immune response to viruses. RNA interference (RNAi) is a technique used for transgene-mediated gene silencing based on the mechanism of posttranscriptional gene silencing (PTGS). 3Physics II Institute, University of Giessen, Giessen, Germany.2Institute of Cellular and Molecular Botany, University of Bonn, Bonn, Germany.1Leibniz Institute of Plant Genetics and Crop Plant Research, Seeland, Germany.Stefanie Lück 1, Tino Kreszies 2, Marc Strickert 3, Patrick Schweizer 1†, Markus Kuhlmann 1 and Dimitar Douchkov 1*
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