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 & PRIME; 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.

Automated summary

In this study, the researchers improved the efficiency of gene silencing by modifying miR168a structure in Arabidopsis thaliana and using tandem two-hit amiRNAs. They demonstrated the effectiveness of this approach in silencing genes involved in miRNA, tasiRNA, and hormone signalling pathways. The authors also compared two-hit amiRNA technology with CRISPR/Cas9 and provided a web-based amiRNA designer for easy design and wide application.