Sachin Teotia, Xiaoran Wang, Na Zhou, Mengmeng Wang, Haiping Liu, Jun Qin, Dianwei Han, Chingwen Li, Christine E. Li, Shangjin Pan, Haifeng Tang, Wenjun Kang, Zhanhui Zhang, Xiaoqing Tang, Ting Peng, Guiliang Tang

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

  • Plant Science
  • Agronomy and Crop Science
  • Biotechnology
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SummaryMicroRNAs (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.

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