A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop/duplex with tandem asymmetrical amiRNA duplexes that follow the statistical rules of miRNA secondary structures. These tandem amiRNA duplexes, called "two-hit" amiRNAs, were shown to have a higher efficiency in silencing GFP and endogenous PDS reporter genes compared to traditional "one-hit" amiRNAs. The authors also demonstrated the effectiveness of "two-hit" amiRNAs in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways, individually or in families. Importantly, "two-hit" amiRNAs were also able to over-express endogenous miRNAs for their functions. The authors compare "two-hit" amiRNA technology with CRISPR/Cas9 and provide a web-based amiRNA designer for easy design and wide application in plants and even animals.

Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis.

MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals.