Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique
Abstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic man...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-06-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-018-1170-4 |
| _version_ | 1811257824029704192 |
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| author | Bin Xiong Zhongkang Li Li Liu Dongdong Zhao Xueli Zhang Changhao Bi |
| author_facet | Bin Xiong Zhongkang Li Li Liu Dongdong Zhao Xueli Zhang Changhao Bi |
| author_sort | Bin Xiong |
| collection | DOAJ |
| description | Abstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. Results An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion. |
| first_indexed | 2024-04-12T18:03:32Z |
| format | Article |
| id | doaj.art-af6d8195c3d04342aa590e457cd118b1 |
| institution | Directory Open Access Journal |
| issn | 1754-6834 |
| language | English |
| last_indexed | 2024-04-12T18:03:32Z |
| publishDate | 2018-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels |
| spelling | doaj.art-af6d8195c3d04342aa590e457cd118b12022-12-22T03:22:04ZengBMCBiotechnology for Biofuels1754-68342018-06-011111910.1186/s13068-018-1170-4Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 techniqueBin Xiong0Zhongkang Li1Li Liu2Dongdong Zhao3Xueli Zhang4Changhao Bi5University of Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesTianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract Background Ralstonia eutropha is an important bacterium for the study of polyhydroxyalkanoates (PHAs) synthesis and CO2 fixation, which makes it a potential strain for industrial PHA production and attractive host for CO2 conversion. Although the bacterium is not recalcitrant to genetic manipulation, current methods for genome editing based on group II introns or single crossover integration of a suicide plasmid are inefficient and time-consuming, which limits the genetic engineering of this organism. Thus, developing an efficient and convenient method for R. eutropha genome editing is imperative. Results An efficient genome editing method for R. eutropha was developed using an electroporation-based CRISPR-Cas9 technique. In our study, the electroporation efficiency of R. eutropha was found to be limited by its restriction-modification (RM) systems. By searching the putative RM systems in R. eutropha H16 using REBASE database and comparing with that in E. coli MG1655, five putative restriction endonuclease genes which are related to the RM systems in R. eutropha were predicated and disrupted. It was found that deletion of H16_A0006 and H16_A0008-9 increased the electroporation efficiency 1658 and 4 times, respectively. Fructose was found to reduce the leaky expression of the arabinose-inducible pBAD promoter, which was used to optimize the expression of cas9, enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha. A total of five genes were edited with efficiencies ranging from 78.3 to 100%. The CRISPR-Cpf1 system and the non-homologous end joining mechanism were also investigated, but failed to yield edited strains. Conclusions We present the first genome editing method for R. eutropha using an electroporation-based CRISPR-Cas9 approach, which significantly increased the efficiency and decreased time to manipulate this facultative chemolithoautotrophic microbe. The novel technique will facilitate more advanced researches and applications of R. eutropha for PHA production and CO2 conversion.http://link.springer.com/article/10.1186/s13068-018-1170-4Ralstonia eutrophaCupriavidus necatorElectroporationCRISPRCas9Genome editing |
| spellingShingle | Bin Xiong Zhongkang Li Li Liu Dongdong Zhao Xueli Zhang Changhao Bi Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique Biotechnology for Biofuels Ralstonia eutropha Cupriavidus necator Electroporation CRISPR Cas9 Genome editing |
| title | Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique |
| title_full | Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique |
| title_fullStr | Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique |
| title_full_unstemmed | Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique |
| title_short | Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique |
| title_sort | genome editing of ralstonia eutropha using an electroporation based crispr cas9 technique |
| topic | Ralstonia eutropha Cupriavidus necator Electroporation CRISPR Cas9 Genome editing |
| url | http://link.springer.com/article/10.1186/s13068-018-1170-4 |
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