The Effect of Dicer Knockout on RNA Interference Using Various Dicer Substrate Small Interfering RNA (DsiRNA) Structures

Small interfering RNAs (siRNAs) are artificial molecules used to silence genes of interest through the RNA interference (RNAi) pathway, mediated by the endoribonuclease Dicer. Dicer-substrate small interfering RNAs (DsiRNAs) are an alternative to conventional 21-mer siRNAs, with an increased effectiveness of up to 100-fold compared to traditional 21-mer designs. DsiRNAs have a novel asymmetric design that allows them to be processed by Dicer into the desired conventional siRNAs. DsiRNAs are a useful tool for sequence-specific gene silencing, but the molecular mechanism underlying their increased efficacy is not precisely understood. In this study, to gain a deeper understanding of Dicer function in DsiRNAs, we designed nicked DsiRNAs with and without tetra-loops to target a specific mRNA sequence, established a Dicer knockout in the HCT116 cell line, and analyzed the efficacy of various DsiRNAs on RNAi-mediated gene silencing activity. The gene silencing activity of all DsiRNAs was reduced in Dicer knockout cells. We demonstrated that tetra-looped DsiRNAs exhibited increased efficacy for gene silencing, which was mediated by Dicer protein. Thus, this study improves our understanding of Dicer function, a key component of RNAi silencing, which will inform RNAi research and applications.