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...

Descripción completa

Detalles Bibliográficos
Autores: Perroud, Pierre-François, Guyon-Debast, Anouchka|||0000-0003-3489-1035, Casacuberta, Josep M.|||0000-0002-5609-4152, Paul, Wyatt, Pichon, Jean-Philippe, Comeau, David, Nogué, Fabien|||0000-0003-0619-4638
Tipo de recurso: artículo
Fecha de publicación:2023
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:309139
Acceso en línea:https://ddd.uab.cat/record/309139
https://dx.doi.org/urn:doi:10.1093/jxb/erad189
Access Level:acceso abierto
Palabra clave:EpegRNA
Genome editing
Physcomitrium patens
Prime editing
Pseudoknot
Split prime editing
id ES_dc180be2bb47deeb38d3cff8c8626dca
oai_identifier_str oai:ddd.uab.cat:309139
network_acronym_str ES
network_name_str España
repository_id_str
spelling Improved prime editing allows for routine predictable gene editing in Physcomitrium patensPerroud, Pierre-FrançoisGuyon-Debast, Anouchka|||0000-0003-3489-1035Casacuberta, Josep M.|||0000-0002-5609-4152Paul, WyattPichon, Jean-PhilippeComeau, DavidNogué, Fabien|||0000-0003-0619-4638EpegRNAGenome 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. 22023-01-0120232023-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/309139https://dx.doi.org/urn:doi:10.1093/jxb/erad189reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengopen accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:3091392026-06-06T12:50:31Z
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|||0000-0003-3489-1035
Casacuberta, Josep M.|||0000-0002-5609-4152
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien|||0000-0003-0619-4638
author Perroud, Pierre-François
author_facet Perroud, Pierre-François
Guyon-Debast, Anouchka|||0000-0003-3489-1035
Casacuberta, Josep M.|||0000-0002-5609-4152
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien|||0000-0003-0619-4638
author_role author
author2 Guyon-Debast, Anouchka|||0000-0003-3489-1035
Casacuberta, Josep M.|||0000-0002-5609-4152
Paul, Wyatt
Pichon, Jean-Philippe
Comeau, David
Nogué, Fabien|||0000-0003-0619-4638
author2_role author
author
author
author
author
author
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 2
2023-01-01
2023
2023-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/309139
https://dx.doi.org/urn:doi:10.1093/jxb/erad189
url https://ddd.uab.cat/record/309139
https://dx.doi.org/urn:doi:10.1093/jxb/erad189
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869421736816017408
score 15,81155

Similares en LA Referencia