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Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two
BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advant...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10053088/ https://www.ncbi.nlm.nih.gov/pubmed/36978193 http://dx.doi.org/10.1186/s13007-023-01010-4 |
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author | Ordon, Jana Kiel, Niklas Becker, Dieter Kretschmer, Carola Schulze-Lefert, Paul Stuttmann, Johannes |
author_facet | Ordon, Jana Kiel, Niklas Becker, Dieter Kretschmer, Carola Schulze-Lefert, Paul Stuttmann, Johannes |
author_sort | Ordon, Jana |
collection | PubMed |
description | BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-023-01010-4. |
format | Online Article Text |
id | pubmed-10053088 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-100530882023-03-30 Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two Ordon, Jana Kiel, Niklas Becker, Dieter Kretschmer, Carola Schulze-Lefert, Paul Stuttmann, Johannes Plant Methods Methodology BACKGROUND: In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus pyogenes (SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated. RESULTS: We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal segment deletions. CONCLUSIONS: Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13007-023-01010-4. BioMed Central 2023-03-28 /pmc/articles/PMC10053088/ /pubmed/36978193 http://dx.doi.org/10.1186/s13007-023-01010-4 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Ordon, Jana Kiel, Niklas Becker, Dieter Kretschmer, Carola Schulze-Lefert, Paul Stuttmann, Johannes Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title | Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title_full | Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title_fullStr | Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title_full_unstemmed | Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title_short | Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two |
title_sort | targeted gene deletion with spcas9 and multiple guide rnas in arabidopsis thaliana: four are better than two |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10053088/ https://www.ncbi.nlm.nih.gov/pubmed/36978193 http://dx.doi.org/10.1186/s13007-023-01010-4 |
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