Dataset Information

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

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.

SUBMITTER: Teotia S

PROVIDER: S-EPMC10440985 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Publications

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

Teotia Sachin S Wang Xiaoran X Zhou Na N Wang Mengmeng M Liu Haiping H Qin Jun J Han Dianwei D Li Chingwen C Li Christine E CE Pan Shangjin S Tang Haifeng H Kang Wenjun W Zhang Zhanhui Z Tang Xiaoqing X Peng Ting T Tang Guiliang G

Plant biotechnology journal 20230701 9

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 pos ...[more]

PMID: 37392408

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Project description:Artificial small RNAs (art-sRNAs) are 21-nucleotide small RNAs (sRNAs) computationally designed to silence plant genes or pathogenic RNAs with high efficacy and specificity. They are typically produced in transgenic plants to induce silencing at the whole-organism level, although their expression in selected tissues for inactivating genes in distal tissues has not been reported. Here, art-sRNAs designed against the magnesium chelatase subunit CHLI-encoding SULFUR gene (NbSu) were agroinfiltrated in Nicotiana benthamiana leaves, and the induction of local and systemic silencing was analyzed phenotypically by monitoring the appearance of the characteristic bleached phenotype, as well as molecularly by analyzing art-sRNA processing, accumulation and targeting activity and efficacy. We found that the two classes of art-sRNAs, artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are able to induce systemic silencing of NbSu, which requires high art-sRNA expression in the vicinity of the leaf petiole but is independent on the production of secondary sRNAs from NbSu mRNAs. Moreover, we revealed that 21-nucleotide amiRNA and syn-tasiRNA duplexes, and not their precursors, are the molecules moving between cells and through the phloem to systemically silence NbSu in upper leaves. In sum, our results indicate that 21-nucleotide art-sRNAs can move throughout the plant to silence plant genes in tissues different from where they are produced. This highlights the biotechnological potential of art-sRNAs, which might be applied locally for triggering whole-plant and highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops. The present study demonstrates that artificial small RNAs, such as artificial microRNAs and synthetic trans-acting small interfering RNAs, can move long distances in plants as 21-nucleotide duplexes, specifically silencing endogenous genes in tissues different from where they are applied. This highlights the biotechnological potential of artificial small RNAs, which might be applied locally for triggering whole-plant, highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops.

Dataset Information

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

Publications

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

Similar Datasets

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