Abstract
RNA interference (RNAi) designates sequence-specific mRNA degradation mediated by small RNAs generated from long double-stranded RNA (dsRNA) by RNase III Dicer. RNAi appears inactive in mammalian cells except for mouse oocytes, where high RNAi activity exists because of an N-terminally truncated Dicer isoform, denoted DicerO. DicerO processes dsRNA into small RNAs more efficiently than the full-length Dicer expressed in somatic cells. DicerO is expressed from an oocyte-specific promoter of retrotransposon origin, which is silenced in other cell types. In this work, we evaluated CRISPR-based strategies for epigenetic targeting of the endogenous Dicer gene to restore DicerO expression and, consequently, RNAi. We show that reactivation of DicerO expression can be achieved in mouse embryonic stem cells, but it is not sufficient to establish a robust canonical RNAi response.
Highlights
Canonical RNA interference (RNAi) has been defined as sequence-specific RNA degradation induced by long double-stranded RNA [1]
To test whether deactivated Cas9” (dCas9)-mediated transcriptional activation could induce DicerO expression from the Dicer gene in mouse embryonic stem cells and fibroblasts, we examined several different dCas9-transcriptional activation designs and obtained the best DicerO activation with a system consisting of dCas9-VP64 enhanced with an MS2-p65-HSF1 module originally developed by Konermann et al [23]
Our results suggest that induction of RNAi in mouse cells may be possible by inducing DicerO expression, but achieving robust RNAi activity requires further optimization of DicerO induction, presumably combined with a strategy reducing the inhibitory effects of innate immunity factors
Summary
Canonical RNA interference (RNAi) has been defined as sequence-specific RNA degradation induced by long double-stranded RNA (dsRNA) [1]. The only known mammalian cell type where RNAi is highly active and functionally important, express a unique, naturally N-terminally truncated Dicer isoform, denoted DicerO (Figure 1A) [14] This truncated isoform arose upon intronic insertion of a long terminal repeat (LTR) from the MTC retrotransposon subfamily, which provides an oocyte-specific promoter and the first exon of DicerO (Figure 1B). To test whether dCas9-mediated transcriptional activation could induce DicerO expression from the Dicer gene in mouse embryonic stem cells and fibroblasts, we examined several different dCas9-transcriptional activation designs and obtained the best DicerO activation with a system consisting of dCas9-VP64 enhanced with an MS2-p65-HSF1 module originally developed by Konermann et al [23] In this system, sgRNA loops protruding from the ribonucleoprotein complex carry a minimal hairpin aptamer, which is bound by the MS2 protein that can be fused with an additional factor enhancing transcription [22,23]. Our results suggest that induction of RNAi in mouse cells may be possible by inducing DicerO expression, but achieving robust RNAi activity requires further optimization of DicerO induction, presumably combined with a strategy reducing the inhibitory effects of innate immunity factors
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