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Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a

Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas n...

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Autores principales: Huang, Teng‐Kuei, Armstrong, Brittney, Schindele, Patrick, Puchta, Holger
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313123/
https://www.ncbi.nlm.nih.gov/pubmed/33511745
http://dx.doi.org/10.1111/pbi.13546
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author Huang, Teng‐Kuei
Armstrong, Brittney
Schindele, Patrick
Puchta, Holger
author_facet Huang, Teng‐Kuei
Armstrong, Brittney
Schindele, Patrick
Puchta, Holger
author_sort Huang, Teng‐Kuei
collection PubMed
description Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site‐specific double‐strand breaks to enhance homologous recombination‐mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas‐based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature‐insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium‐mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T‐DNA integration. Thus, we were not only able to obtain CRISPR/Cas‐based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.
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spelling pubmed-83131232021-07-31 Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a Huang, Teng‐Kuei Armstrong, Brittney Schindele, Patrick Puchta, Holger Plant Biotechnol J Research Articles Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site‐specific double‐strand breaks to enhance homologous recombination‐mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas‐based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature‐insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium‐mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T‐DNA integration. Thus, we were not only able to obtain CRISPR/Cas‐based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops. John Wiley and Sons Inc. 2021-01-28 2021-07 /pmc/articles/PMC8313123/ /pubmed/33511745 http://dx.doi.org/10.1111/pbi.13546 Text en © 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Research Articles
Huang, Teng‐Kuei
Armstrong, Brittney
Schindele, Patrick
Puchta, Holger
Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title_full Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title_fullStr Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title_full_unstemmed Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title_short Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a
title_sort efficient gene targeting in nicotiana tabacum using crispr/sacas9 and temperature tolerant lbcas12a
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8313123/
https://www.ncbi.nlm.nih.gov/pubmed/33511745
http://dx.doi.org/10.1111/pbi.13546
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