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CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion

BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some pr...

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Detalles Bibliográficos
Autores principales: Liu, Chunyan, Yue, Yaxin, Xue, Yanfen, Zhou, Cheng, Ma, Yanhe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10576340/
https://www.ncbi.nlm.nih.gov/pubmed/37838676
http://dx.doi.org/10.1186/s12934-023-02214-y
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author Liu, Chunyan
Yue, Yaxin
Xue, Yanfen
Zhou, Cheng
Ma, Yanhe
author_facet Liu, Chunyan
Yue, Yaxin
Xue, Yanfen
Zhou, Cheng
Ma, Yanhe
author_sort Liu, Chunyan
collection PubMed
description BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter P(ara) for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-023-02214-y.
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spelling pubmed-105763402023-10-15 CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion Liu, Chunyan Yue, Yaxin Xue, Yanfen Zhou, Cheng Ma, Yanhe Microb Cell Fact Research BACKGROUND: Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis. RESULTS: Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction between the HR and the NHEJ repair system, were further investigated. Finally, the optimized CRISPR-Cas9-NHEJ editing system was able to delete DNA fragments up to 50 kb rapidly with high efficiency of 31.3%, when three sgRNAs on the Crick/Watson/Watson DNA double-strands and the arabinose-induced promoter P(ara) for Cas9 were used, along with the background expression of the HR repair system. CONCLUSIONS: This was the first report of CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas spp. These results not only suggest that this editing system is a powerful genome engineering tool for constructing chassis cells in Halomonas, but also extend the application of the NHEJ repair system. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-023-02214-y. BioMed Central 2023-10-14 /pmc/articles/PMC10576340/ /pubmed/37838676 http://dx.doi.org/10.1186/s12934-023-02214-y Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Liu, Chunyan
Yue, Yaxin
Xue, Yanfen
Zhou, Cheng
Ma, Yanhe
CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title_full CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title_fullStr CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title_full_unstemmed CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title_short CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion
title_sort crispr-cas9 assisted non-homologous end joining genome editing system of halomonas bluephagenesis for large dna fragment deletion
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10576340/
https://www.ncbi.nlm.nih.gov/pubmed/37838676
http://dx.doi.org/10.1186/s12934-023-02214-y
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