Improved prime editing allows for routine predictable gene editing in Physcomitrium patens

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its...

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Authors: Perroud, Pierre-François, Guyon-Debast, Anouchka, Casacuberta, Josep M., Paul, Wyatt, Pichon, Jean-Philippe, Comeau, David, Nogué, Fabien
Format: article
Status:Published version
Publication Date:2023
Country:España
Institution:Consejo Superior de Investigaciones Científicas (CSIC)
Repository:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/343256
Online Access:http://hdl.handle.net/10261/343256
Access Level:Open access
Keyword:EpegRNA
Genome editing
Physcomitrium patens
Prime editing
Pseudoknot
Split prime editing
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repository_id_str
spelling Improved prime editing allows for routine predictable gene editing in Physcomitrium patensPerroud, Pierre-FrançoisGuyon-Debast, AnouchkaCasacuberta, Josep M.Paul, WyattPichon, Jean-PhilippeComeau, DavidNogué, FabienEpegRNAGenome editingPhyscomitrium patensPrime editingPseudoknotSplit prime editingEfficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3ʹ extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.This study was supported by Limagrain Europe via the CRISPLUS project. The Institut Jean-Pierre Bourgin benefits from the support of the LabEx Saclay Plant Sciences-SPS (Agence Nationale de la Recherche (ANR)-10-LABX-0040-SPS).Peer reviewedSociety for Experimental BiologyLimagrain EuropeAgence Nationale de la Recherche (France)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202420242023info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://hdl.handle.net/10261/343256reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)InglésThe underlying dataset has been published as supplementary material of the article in the publisher platform at https://doi.org/10.1093/jxb/erad189https://doi.org/10.1093/jxb/erad189Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3432562026-05-22T06:33:51Z
dc.title.none.fl_str_mv Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
title Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
spellingShingle Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
Perroud, Pierre-François
EpegRNA
Genome editing
Physcomitrium patens
Prime editing
Pseudoknot
Split prime editing
title_short Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
title_full Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
title_fullStr Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
title_full_unstemmed Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
title_sort Improved prime editing allows for routine predictable gene editing in Physcomitrium patens
dc.creator.none.fl_str_mv Perroud, Pierre-François
Guyon-Debast, Anouchka
Casacuberta, Josep M.
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien
author Perroud, Pierre-François
author_facet Perroud, Pierre-François
Guyon-Debast, Anouchka
Casacuberta, Josep M.
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien
author_role author
author2 Guyon-Debast, Anouchka
Casacuberta, Josep M.
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Limagrain Europe
Agence Nationale de la Recherche (France)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv EpegRNA
Genome editing
Physcomitrium patens
Prime editing
Pseudoknot
Split prime editing
topic EpegRNA
Genome editing
Physcomitrium patens
Prime editing
Pseudoknot
Split prime editing
description Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements. Using a standardized protoplast transfection procedure, multiple prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3ʹ extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.
publishDate 2023
dc.date.none.fl_str_mv 2023
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/343256
url http://hdl.handle.net/10261/343256
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv The underlying dataset has been published as supplementary material of the article in the publisher platform at https://doi.org/10.1093/jxb/erad189
https://doi.org/10.1093/jxb/erad189
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Society for Experimental Biology
publisher.none.fl_str_mv Society for Experimental Biology
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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