A Strategy to Use Artificial MicroRNAs for Gene Silencing and Overcome (AmiGO)

Essential genes of multicellular organisms encode products that are indispensable for proper development, reproduction, and survival of the organism. Knocking out essential genes often causes organismal death, which challenges the study of the role of these genes in traits. Here we conduct an Artificial MicroRNAs for Gene silencing and Overcome (AmiGO) strategy, using artificial microRNAs to generate cell type-specific loss-of-function mutants, to unravel cellular functions of essential genes in Arabidopsis thaliana (At). This strategy allows genetic complementation using wild-type genomic fragments or coding sequences (CDS). As a proof of concept for the AmiGO strategy, we describe its application to the gametophytic-lethal gene of RETINOBLASTOMA-RELATED (RBR). Mature AmiGO-RBR, which targets the three prime untranslated regions (3'UTR) of RBR messenger RNA (mRNA), was cloned into the backbone of AtmiR319a precursor. To dissect RBR functions in different cell types, we used tissue-specific promoters to drive the expression of AmiGO-RBR precursor, thereby repressing RBR expression in the corresponding tissues. By developing an AmiGO-RBR-sensor line, we showed that the AmiGO-RBR target gene silencing is efficient and tissue-specific. Moreover, since we were able to complement the AmiGO-RBR-induced phenotypes by expressing a RBR genomic fragment that lacks its mRNA 3'UTR region, we demonstrated that AmiGO-RBR silences its target in a sequence-specific manner. Although tested in Arabidopsis, this strategy can be applied to any plant or animal models, for which an endogenous precursor miRNA has been cloned.