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ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana

Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the...

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Autores principales: Wang, Zupeng, Liu, Xiaoying, Xie, Xiaodong, Deng, Lei, Zheng, Hao, Pan, Hui, Li, Dawei, Li, Li, Zhong, Caihong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198424/
https://www.ncbi.nlm.nih.gov/pubmed/34073486
http://dx.doi.org/10.3390/ijms22115663
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author Wang, Zupeng
Liu, Xiaoying
Xie, Xiaodong
Deng, Lei
Zheng, Hao
Pan, Hui
Li, Dawei
Li, Li
Zhong, Caihong
author_facet Wang, Zupeng
Liu, Xiaoying
Xie, Xiaodong
Deng, Lei
Zheng, Hao
Pan, Hui
Li, Dawei
Li, Li
Zhong, Caihong
author_sort Wang, Zupeng
collection PubMed
description Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants.
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spelling pubmed-81984242021-06-14 ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana Wang, Zupeng Liu, Xiaoying Xie, Xiaodong Deng, Lei Zheng, Hao Pan, Hui Li, Dawei Li, Li Zhong, Caihong Int J Mol Sci Article Adenine base editor containing TadA8e (ABE8e) has been reported in rice. However, the application of ABE8e in other plant species has not been described, and the comparison between ABE8e and ABE7.10, which is widely used in plants, has also been poorly studied. Here, we developed the ABE8e with the polycistronic tRNA-gRNA expression cassette (PTG-ABE8e) and PTG-ABE7.10 and compared their A-to-G editing efficiencies using both transient and stable transformation in the allotetraploid Nicotiana benthamiana. We found that the editing efficiency of PTG-ABE8e was significantly higher than that of PTG-ABE7.10, indicating that ABE8e was more efficient for A-to-G conversion in N. benthamiana. We further optimized the ABE8e editing efficiency by changing the sgRNA expression cassette and demonstrated that both PTG and single transcript unit (STU) enhanced ABE8e efficiency for A-to-G conversion in N. benthamiana. We also estimated the potential off-target effect of PTG-ABE8e at potential off-targeting sites predicted using an online tool in transgenic plants, and no off-target editing event was found for potential off-targeting sites selected, indicating that ABE8e could specifically facilitate A-to-G conversion. Our results showed that ABE8e with PTG structure was more suitable for A-to-G conversion in N. benthamiana and provided valuable clues for optimizing ABE tools in other plants. MDPI 2021-05-26 /pmc/articles/PMC8198424/ /pubmed/34073486 http://dx.doi.org/10.3390/ijms22115663 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wang, Zupeng
Liu, Xiaoying
Xie, Xiaodong
Deng, Lei
Zheng, Hao
Pan, Hui
Li, Dawei
Li, Li
Zhong, Caihong
ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title_full ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title_fullStr ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title_full_unstemmed ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title_short ABE8e with Polycistronic tRNA-gRNA Expression Cassette Sig-Nificantly Improves Adenine Base Editing Efficiency in Nicotiana benthamiana
title_sort abe8e with polycistronic trna-grna expression cassette sig-nificantly improves adenine base editing efficiency in nicotiana benthamiana
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8198424/
https://www.ncbi.nlm.nih.gov/pubmed/34073486
http://dx.doi.org/10.3390/ijms22115663
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