Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1

[EN]CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions...

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Detalhes bibliográficos
Autores: Jiménez García, Alberto, Hoff, Birgit, Revuelta Doval, José Luis
Tipo de documento: artigo
Estado:Versão publicada
Data de publicação:2020
País:España
Recursos:Universidad de Salamanca (USAL)
Repositório:GREDOS. Repositorio Institucional de la Universidad de Salamanca
OAI Identifier:oai:gredos.usal.es:10366/150744
Acesso em linha:http://hdl.handle.net/10366/150744
Access Level:Acceso aberto
Palavra-chave:Ashbya gossypii
CRISPR/Cpf1
Genome engineering
multiplex gene editing
Filamentous fungi
Multiplex gene editing
2409.02 Ingeniería Genética
2414.06 Hongos
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oai_identifier_str oai:gredos.usal.es:10366/150744
network_acronym_str ES
network_name_str España
repository_id_str
spelling Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1Jiménez García, AlbertoHoff, BirgitRevuelta Doval, José LuisAshbya gossypiiCRISPR/Cpf1Genome engineeringmultiplex gene editingFilamentous fungiAshbya gossypiiCRISPR/Cpf1Genome engineeringMultiplex gene editingFilamentous fungi2409.02 Ingeniería Genética2414.06 Hongos[EN]CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific5′-NGG-3′ PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5′-TTTN-3′)to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multiCRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.202220222020info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10366/150744reponame:GREDOS. Repositorio Institucional de la Universidad de Salamancainstname:Universidad de Salamanca (USAL)InglésBIO2017-88435-RSA016P17Attribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:gredos.usal.es:10366/1507442026-06-07T06:28:51Z
dc.title.none.fl_str_mv Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
title Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
spellingShingle Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
Jiménez García, Alberto
Ashbya gossypii
CRISPR/Cpf1
Genome engineering
multiplex gene editing
Filamentous fungi
Ashbya gossypii
CRISPR/Cpf1
Genome engineering
Multiplex gene editing
Filamentous fungi
2409.02 Ingeniería Genética
2414.06 Hongos
title_short Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
title_full Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
title_fullStr Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
title_full_unstemmed Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
title_sort Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1
dc.creator.none.fl_str_mv Jiménez García, Alberto
Hoff, Birgit
Revuelta Doval, José Luis
author Jiménez García, Alberto
author_facet Jiménez García, Alberto
Hoff, Birgit
Revuelta Doval, José Luis
author_role author
author2 Hoff, Birgit
Revuelta Doval, José Luis
author2_role author
author
dc.subject.none.fl_str_mv Ashbya gossypii
CRISPR/Cpf1
Genome engineering
multiplex gene editing
Filamentous fungi
Ashbya gossypii
CRISPR/Cpf1
Genome engineering
Multiplex gene editing
Filamentous fungi
2409.02 Ingeniería Genética
2414.06 Hongos
topic Ashbya gossypii
CRISPR/Cpf1
Genome engineering
multiplex gene editing
Filamentous fungi
Ashbya gossypii
CRISPR/Cpf1
Genome engineering
Multiplex gene editing
Filamentous fungi
2409.02 Ingeniería Genética
2414.06 Hongos
description [EN]CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific5′-NGG-3′ PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5′-TTTN-3′)to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multiCRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.
publishDate 2020
dc.date.none.fl_str_mv 2020
2022
2022
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10366/150744
url http://hdl.handle.net/10366/150744
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv BIO2017-88435-R
SA016P17
dc.rights.none.fl_str_mv Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Attribution-NonCommercial-NoDerivatives 4.0 Internacional
http://creativecommons.org/licenses/by-nc-nd/4.0/
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:GREDOS. Repositorio Institucional de la Universidad de Salamanca
instname:Universidad de Salamanca (USAL)
instname_str Universidad de Salamanca (USAL)
reponame_str GREDOS. Repositorio Institucional de la Universidad de Salamanca
collection GREDOS. Repositorio Institucional de la Universidad de Salamanca
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,198674

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