Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system
Abstract Background Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein express...
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| Format: | Article |
| Language: | English |
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BMC
2017-11-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-017-0814-6 |
| _version_ | 1828842590726782976 |
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| author | Feng Peng Xinyue Wang Yang Sun Guibin Dong Yankun Yang Xiuxia Liu Zhonghu Bai |
| author_facet | Feng Peng Xinyue Wang Yang Sun Guibin Dong Yankun Yang Xiuxia Liu Zhonghu Bai |
| author_sort | Feng Peng |
| collection | DOAJ |
| description | Abstract Background Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum. |
| first_indexed | 2024-12-12T20:27:47Z |
| format | Article |
| id | doaj.art-6c51444e24c54f2bada94def90183b2c |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-12T20:27:47Z |
| publishDate | 2017-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-6c51444e24c54f2bada94def90183b2c2022-12-22T00:13:06ZengBMCMicrobial Cell Factories1475-28592017-11-0116111310.1186/s12934-017-0814-6Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 systemFeng Peng0Xinyue Wang1Yang Sun2Guibin Dong3Yankun Yang4Xiuxia Liu5Zhonghu Bai6National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityNational Engineering Laboratory for Cereal Fermentation Technology, Jiangnan UniversityAbstract Background Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.http://link.springer.com/article/10.1186/s12934-017-0814-6CRISPR/Cas9Corynebacterium glutamicumGenome editingProtein expression |
| spellingShingle | Feng Peng Xinyue Wang Yang Sun Guibin Dong Yankun Yang Xiuxia Liu Zhonghu Bai Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system Microbial Cell Factories CRISPR/Cas9 Corynebacterium glutamicum Genome editing Protein expression |
| title | Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system |
| title_full | Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system |
| title_fullStr | Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system |
| title_full_unstemmed | Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system |
| title_short | Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system |
| title_sort | efficient gene editing in corynebacterium glutamicum using the crispr cas9 system |
| topic | CRISPR/Cas9 Corynebacterium glutamicum Genome editing Protein expression |
| url | http://link.springer.com/article/10.1186/s12934-017-0814-6 |
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