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Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing
Year:
2022
Source of publication :
Plant Cell Reports
Authors :
Bocobza, Samuel
;
.
Kumar, Manoj
;
.
Volume :
Co-Authors:
  • Manoj Kumar, 
  • Pankaj Kumar Tripathi, 
  • Dana Ayzenshtat, 
  • Adar Marko, 
  • Zohar Forotan 
  • Samuel E. Bocobza
Facilitators :
From page:
0
To page:
0
(
Total pages:
1
)
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.

Note:
Related Files :
ARGAUNOTE7
betalain
gene editing
gene silencing
NtSGS3
PDS
Plant transformation
PTGS
RFP
RNAi
Show More
Related Content
More details
DOI :
10.1007/s00299-022-02903-9
Article number:
0
Affiliations:
Database:
Scopus
Publication Type:
article
;
.
Language:
English
Editors' remarks:
ID:
60838
Last updated date:
01/08/2022 17:28
Creation date:
01/08/2022 17:28
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Scientific Publication
Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing
  • Manoj Kumar, 
  • Pankaj Kumar Tripathi, 
  • Dana Ayzenshtat, 
  • Adar Marko, 
  • Zohar Forotan 
  • Samuel E. Bocobza
Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing

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.

Scientific Publication
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