Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata

  • Oh, Youngbin (Department of Biological Sciences, Korea Advanced Institute for Science and Technology) ;
  • Kim, Sang-Gyu (Department of Biological Sciences, Korea Advanced Institute for Science and Technology)
  • Received : 2021.09.13
  • Accepted : 2021.09.30
  • Published : 2021.12.31
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Abstract

The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M1 progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

Keywords

Acknowledgement

We thank Emily Wheeler, for editorial assistance and Joohee Kim, for technical assistance. The targeted deep sequencing was supported by Bio Core facilities in KAIST. Y.O. and S.G.K. have filed a patent application based on this work. This work was supported by KAIST-funded Global Singularity Research Program (N11200021) and the Samsung Science & Technology Foundation (SSTF-BA1901-10).

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