Easy‐Curing and pH‐Regulated CRISPR‐Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris
ABSTRACT 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, exp...
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Wiley
2024-12-01
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| Series: | Microbial Biotechnology |
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| Online Access: | https://doi.org/10.1111/1751-7915.70060 |
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| author | Javier Nicolás Garay‐Novillo José Ángel Ruiz‐Masó Gloria delSolar José Luis Barra |
| author_facet | Javier Nicolás Garay‐Novillo José Ángel Ruiz‐Masó Gloria delSolar José Luis Barra |
| author_sort | Javier Nicolás Garay‐Novillo |
| collection | DOAJ |
| description | ABSTRACT 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. |
| format | Article |
| id | doaj-art-b00902a293864cb6a10fa149dac2ef65 |
| institution | DOAJ |
| issn | 1751-7915 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj-art-b00902a293864cb6a10fa149dac2ef652025-08-20T02:50:47ZengWileyMicrobial Biotechnology1751-79152024-12-011712n/an/a10.1111/1751-7915.70060Easy‐Curing and pH‐Regulated CRISPR‐Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremorisJavier Nicolás Garay‐Novillo0José Ángel Ruiz‐Masó1Gloria delSolar2José Luis Barra3Departamento de Química Biológica Ranwel Caputto, CIQUIBIC‐CONICET, Facultad de Ciencias Químicas Universidad Nacional de Córdoba Córdoba ArgentinaDepartamento de Biotecnología Microbiana y de Plantas Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas Madrid SpainDepartamento de Biotecnología Microbiana y de Plantas Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas Madrid SpainDepartamento de Química Biológica Ranwel Caputto, CIQUIBIC‐CONICET, Facultad de Ciencias Químicas Universidad Nacional de Córdoba Córdoba ArgentinaABSTRACT 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.https://doi.org/10.1111/1751-7915.70060CRISPR‐Cas9gene‐editingLactococcus cremorisplasmid curingplasmid stability |
| spellingShingle | Javier Nicolás Garay‐Novillo José Ángel Ruiz‐Masó Gloria delSolar José Luis Barra Easy‐Curing and pH‐Regulated CRISPR‐Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris Microbial Biotechnology CRISPR‐Cas9 gene‐editing Lactococcus cremoris plasmid curing plasmid stability |
| title | 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_short | 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 |
| topic | CRISPR‐Cas9 gene‐editing Lactococcus cremoris plasmid curing plasmid stability |
| url | https://doi.org/10.1111/1751-7915.70060 |
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