Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris

Autores
Garay Novillo, Javier Nicolás; Ruiz Masó, José Ángel; Del Solar, Gloria; Barra, Jose Luis
Año de publicación
2024
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors, based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.
Fil: Garay Novillo, Javier Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina
Fil: Ruiz Masó, José Ángel. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España
Fil: Del Solar, Gloria. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España
Fil: Barra, Jose Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
Materia
Lactococcus cremoris
CRISPR-Cas9
Gene-editing
Plasmid curing
Plasmid stability
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/256588
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/256588
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremorisGaray Novillo, Javier NicolásRuiz Masó, José ÁngelDel Solar, GloriaBarra, Jose LuisLactococcus cremorisCRISPR-Cas9Gene-editingPlasmid curingPlasmid stabilityhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors, based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.Fil: Garay Novillo, Javier Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Ruiz Masó, José Ángel. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Del Solar, Gloria. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; EspañaFil: Barra, Jose Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaJohn Wiley & Sons2024-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/256588Garay Novillo, Javier Nicolás; Ruiz Masó, José Ángel; Del Solar, Gloria; Barra, Jose Luis; Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris; John Wiley & Sons; Microbial Biotechnology; 17; 12; 12-2024; 1-151751-79151751-7915CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70060info:eu-repo/semantics/altIdentifier/doi/10.1111/1751-7915.70060info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2026-06-04T11:03:32Zoai:ri.conicet.gov.ar:11336/256588instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982026-06-04 11:03:32.416CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
title Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
spellingShingle Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
Garay Novillo, Javier Nicolás
Lactococcus cremoris
CRISPR-Cas9
Gene-editing
Plasmid curing
Plasmid stability
title_short Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
title_full Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
title_fullStr Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
title_full_unstemmed Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
title_sort Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris
dc.creator.none.fl_str_mv Garay Novillo, Javier Nicolás
Ruiz Masó, José Ángel
Del Solar, Gloria
Barra, Jose Luis
author Garay Novillo, Javier Nicolás
author_facet Garay Novillo, Javier Nicolás
Ruiz Masó, José Ángel
Del Solar, Gloria
Barra, Jose Luis
author_role author
author2 Ruiz Masó, José Ángel
Del Solar, Gloria
Barra, Jose Luis
author2_role author
author
author
dc.subject.none.fl_str_mv Lactococcus cremoris
CRISPR-Cas9
Gene-editing
Plasmid curing
Plasmid stability
topic Lactococcus cremoris
CRISPR-Cas9
Gene-editing
Plasmid curing
Plasmid stability
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors, based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.
Fil: Garay Novillo, Javier Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina
Fil: Ruiz Masó, José Ángel. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España
Fil: Del Solar, Gloria. Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas; España
Fil: Barra, Jose Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina
description In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors, based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.
publishDate 2024
dc.date.none.fl_str_mv 2024-12
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/256588
Garay Novillo, Javier Nicolás; Ruiz Masó, José Ángel; Del Solar, Gloria; Barra, Jose Luis; Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris; John Wiley & Sons; Microbial Biotechnology; 17; 12; 12-2024; 1-15
1751-7915
1751-7915
CONICET Digital
CONICET
url http://hdl.handle.net/11336/256588
identifier_str_mv Garay Novillo, Javier Nicolás; Ruiz Masó, José Ángel; Del Solar, Gloria; Barra, Jose Luis; Easy-curing and pH-regulated CRISPR-Cas9 plasmids for gene editing and plasmid curing in Lactococcus cremoris; John Wiley & Sons; Microbial Biotechnology; 17; 12; 12-2024; 1-15
1751-7915
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://enviromicro-journals.onlinelibrary.wiley.com/doi/10.1111/1751-7915.70060
info:eu-repo/semantics/altIdentifier/doi/10.1111/1751-7915.70060
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv John Wiley & Sons
publisher.none.fl_str_mv John Wiley & Sons
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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score 12.832306

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