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Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system

BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches...

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Autores principales: Sun, Jun, Wang, Qingzhuo, Jiang, Yu, Wen, Zhiqiang, Yang, Lirong, Wu, Jianping, Yang, Sheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851096/
https://www.ncbi.nlm.nih.gov/pubmed/29534717
http://dx.doi.org/10.1186/s12934-018-0887-x
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author Sun, Jun
Wang, Qingzhuo
Jiang, Yu
Wen, Zhiqiang
Yang, Lirong
Wu, Jianping
Yang, Sheng
author_facet Sun, Jun
Wang, Qingzhuo
Jiang, Yu
Wen, Zhiqiang
Yang, Lirong
Wu, Jianping
Yang, Sheng
author_sort Sun, Jun
collection PubMed
description BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. RESULTS: In this study, we established a fast and convenient CRISPR–Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR–Cas9 system, we also constructed a CRISPR–Cpf1 system, which we validated for genome editing in P. putida KT2440. CONCLUSIONS: In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0887-x) contains supplementary material, which is available to authorized users.
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spelling pubmed-58510962018-03-21 Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system Sun, Jun Wang, Qingzhuo Jiang, Yu Wen, Zhiqiang Yang, Lirong Wu, Jianping Yang, Sheng Microb Cell Fact Research BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a “generally recognized as safe”-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool. RESULTS: In this study, we established a fast and convenient CRISPR–Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR–Cas9 system, we also constructed a CRISPR–Cpf1 system, which we validated for genome editing in P. putida KT2440. CONCLUSIONS: In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440. [Image: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12934-018-0887-x) contains supplementary material, which is available to authorized users. BioMed Central 2018-03-13 /pmc/articles/PMC5851096/ /pubmed/29534717 http://dx.doi.org/10.1186/s12934-018-0887-x Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Sun, Jun
Wang, Qingzhuo
Jiang, Yu
Wen, Zhiqiang
Yang, Lirong
Wu, Jianping
Yang, Sheng
Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title_full Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title_fullStr Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title_full_unstemmed Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title_short Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system
title_sort genome editing and transcriptional repression in pseudomonas putida kt2440 via the type ii crispr system
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5851096/
https://www.ncbi.nlm.nih.gov/pubmed/29534717
http://dx.doi.org/10.1186/s12934-018-0887-x
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