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Evaluation of genome and base editing tools in maize protoplasts

INTRODUCTION: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficienc...

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Autores principales: Fierlej, Yannick, Jacquier, Nathanaël M. A., Guille, Loïc, Just, Jérémy, Montes, Emilie, Richard, Christelle, Loue-Manifel, Jeanne, Depège-Fargeix, Nathalie, Gaillard, Antoine, Widiez, Thomas, Rogowsky, Peter M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9744195/
https://www.ncbi.nlm.nih.gov/pubmed/36518521
http://dx.doi.org/10.3389/fpls.2022.1010030
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author Fierlej, Yannick
Jacquier, Nathanaël M. A.
Guille, Loïc
Just, Jérémy
Montes, Emilie
Richard, Christelle
Loue-Manifel, Jeanne
Depège-Fargeix, Nathalie
Gaillard, Antoine
Widiez, Thomas
Rogowsky, Peter M.
author_facet Fierlej, Yannick
Jacquier, Nathanaël M. A.
Guille, Loïc
Just, Jérémy
Montes, Emilie
Richard, Christelle
Loue-Manifel, Jeanne
Depège-Fargeix, Nathalie
Gaillard, Antoine
Widiez, Thomas
Rogowsky, Peter M.
author_sort Fierlej, Yannick
collection PubMed
description INTRODUCTION: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. METHODS: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. RESULTS AND DISCUSSION: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing.
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spelling pubmed-97441952022-12-13 Evaluation of genome and base editing tools in maize protoplasts Fierlej, Yannick Jacquier, Nathanaël M. A. Guille, Loïc Just, Jérémy Montes, Emilie Richard, Christelle Loue-Manifel, Jeanne Depège-Fargeix, Nathalie Gaillard, Antoine Widiez, Thomas Rogowsky, Peter M. Front Plant Sci Plant Science INTRODUCTION: Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. METHODS: To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. RESULTS AND DISCUSSION: Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing. Frontiers Media S.A. 2022-11-28 /pmc/articles/PMC9744195/ /pubmed/36518521 http://dx.doi.org/10.3389/fpls.2022.1010030 Text en Copyright © 2022 Fierlej, Jacquier, Guille, Just, Montes, Richard, Loue-Manifel, Depège-Fargeix, Gaillard, Widiez and Rogowsky https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Fierlej, Yannick
Jacquier, Nathanaël M. A.
Guille, Loïc
Just, Jérémy
Montes, Emilie
Richard, Christelle
Loue-Manifel, Jeanne
Depège-Fargeix, Nathalie
Gaillard, Antoine
Widiez, Thomas
Rogowsky, Peter M.
Evaluation of genome and base editing tools in maize protoplasts
title Evaluation of genome and base editing tools in maize protoplasts
title_full Evaluation of genome and base editing tools in maize protoplasts
title_fullStr Evaluation of genome and base editing tools in maize protoplasts
title_full_unstemmed Evaluation of genome and base editing tools in maize protoplasts
title_short Evaluation of genome and base editing tools in maize protoplasts
title_sort evaluation of genome and base editing tools in maize protoplasts
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9744195/
https://www.ncbi.nlm.nih.gov/pubmed/36518521
http://dx.doi.org/10.3389/fpls.2022.1010030
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