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Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity

Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), an...

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Autores principales: Biswas, Sudip, Bridgeland, Aya, Irum, Samra, Thomson, Michael J., Septiningsih, Endang M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9456013/
https://www.ncbi.nlm.nih.gov/pubmed/36077206
http://dx.doi.org/10.3390/ijms23179809
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author Biswas, Sudip
Bridgeland, Aya
Irum, Samra
Thomson, Michael J.
Septiningsih, Endang M.
author_facet Biswas, Sudip
Bridgeland, Aya
Irum, Samra
Thomson, Michael J.
Septiningsih, Endang M.
author_sort Biswas, Sudip
collection PubMed
description Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement.
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spelling pubmed-94560132022-09-09 Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity Biswas, Sudip Bridgeland, Aya Irum, Samra Thomson, Michael J. Septiningsih, Endang M. Int J Mol Sci Article Precise editing of the plant genome has long been desired for functional genomic research and crop breeding. Prime editing is a newly developed precise editing technology based on CRISPR-Cas9, which uses an engineered reverse transcriptase (RT), a catalytically impaired Cas9 endonuclease (nCas9), and a prime editing guide RNA (pegRNA). In addition, prime editing has a wider range of editing types than base editing and can produce nearly all types of edits. Although prime editing was first established in human cells, it has recently been applied to plants. As a relatively new technique, optimization will be needed to increase the editing efficiency in different crops. In this study, we successfully edited a mutant GFP in rice, peanut, chickpea, and cowpea protoplasts. In rice, up to 16 times higher editing efficiency was achieved with a dual pegRNA than the single pegRNA containing vectors. Edited-mutant GFP protoplasts have also been obtained in peanut, chickpea, and cowpea after transformation with the dual pegRNA vectors, albeit with much lower editing efficiency than in rice, ranging from 0.2% to 0.5%. These initial results promise to expedite the application of prime editing in legume breeding programs to accelerate crop improvement. MDPI 2022-08-29 /pmc/articles/PMC9456013/ /pubmed/36077206 http://dx.doi.org/10.3390/ijms23179809 Text en © 2022 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
Biswas, Sudip
Bridgeland, Aya
Irum, Samra
Thomson, Michael J.
Septiningsih, Endang M.
Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title_full Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title_fullStr Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title_full_unstemmed Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title_short Optimization of Prime Editing in Rice, Peanut, Chickpea, and Cowpea Protoplasts by Restoration of GFP Activity
title_sort optimization of prime editing in rice, peanut, chickpea, and cowpea protoplasts by restoration of gfp activity
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9456013/
https://www.ncbi.nlm.nih.gov/pubmed/36077206
http://dx.doi.org/10.3390/ijms23179809
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