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A high-efficiency gene silencing in plants using two-hit asymmetrical artificial MicroRNAs |
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| Authors: | 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 |
| Institution: | 1. College of Agronomy, Henan Agricultural University, Zhengzhou, China
Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, USA Department of Biotechnology, Sharda University, Greater Noida, India Equal contribution as first authors.;2. School of Life Sciences, Henan Agricultural University, Zhengzhou, China;3. Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, USA;4. Gene Suppression Laboratory, Department of Plant and Soil Sciences and Kentucky Tobacco and Research Development Center, University of Kentucky, Lexington, Kentucky, USA;5. Department of Computer Science, University of Kentucky, Lexington, Kentucky, USA;6. SQS Lexington Delivery Center, Lexington, Kentucky, USA;7. College of Agronomy, Henan Agricultural University, Zhengzhou, China;8. Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, USA Gene Suppression Laboratory, Department of Plant and Soil Sciences and Kentucky Tobacco and Research Development Center, University of Kentucky, Lexington, Kentucky, USA |
| Abstract: | 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. |
| Keywords: | miRNA artificial miRNAs gene silencing two-hit amiRs Arabidopsis |