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Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing

Plant Cell Reports volume 41, pages 1987–2003 (2022)Cite this article

Abstract

Key message

An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events.

Abstract

Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5′ or 3′ processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.

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Data availability

The data underlying this article are available in the article and in its online supplemental material.

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Acknowledgements

We are very thankful to Dr. Tzahi Arazi for his precious advice and critical reading of this manuscript. We are also grateful to Prof. Asaph Aharoni for providing the pX11 construct (Polturak et al. 2016) used for betalain biosynthesis.

Funding

This study was financially supported by the Chief Scientist—Ministry of Agriculture and Rural Development No. 20-01-0209 as part of the National Center for Genome Editing in Agriculture.

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Authors and Affiliations

  1. Department of Ornamental Plants and Agricultural Biotechnology, The Institute of Plant Sciences, The Volcani Center, ARO, Beit Dagan, Israel

    Manoj Kumar, Pankaj Kumar Tripathi, Dana Ayzenshtat, Adar Marko, Zohar Forotan & Samuel E. Bocobza

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  1. Manoj Kumar
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  2. Pankaj Kumar Tripathi
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  3. Dana Ayzenshtat
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  4. Adar Marko
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  5. Zohar Forotan
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  6. Samuel E. Bocobza
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Contributions

SB conceived and designed the research. MK, PKT, DA, ZF, and AM conducted the experiments. SB supervised the overall work and critically analyzed all the results. MK and SB wrote the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Samuel E. Bocobza.

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The authors declare that they have no conflicts of interest.

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Communicated by Baochun Li.

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Kumar, M., Tripathi, P.K., Ayzenshtat, D. et al. Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing. Plant Cell Rep 41, 1987–2003 (2022). https://doi.org/10.1007/s00299-022-02903-9

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  • DOI: https://doi.org/10.1007/s00299-022-02903-9

Keywords

  • Gene silencing
  • RNAi
  • PTGS
  • NtSGS3
  • RFP
  • PDS
  • ARGAUNOTE7
  • Betalain
  • Plant transformation
  • Gene editing