Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus
Abstract Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the in...
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| Format: | Article |
| Language: | English |
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BMC
2017-08-01
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| Series: | Microbial Cell Factories |
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| Online Access: | http://link.springer.com/article/10.1186/s12934-017-0758-x |
| _version_ | 1811322539416223744 |
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| author | Ying-jie Yang Ye Wang Zhi-feng Li Ya Gong Peng Zhang Wen-chao Hu Duo-hong Sheng Yue-zhong Li |
| author_facet | Ying-jie Yang Ye Wang Zhi-feng Li Ya Gong Peng Zhang Wen-chao Hu Duo-hong Sheng Yue-zhong Li |
| author_sort | Ying-jie Yang |
| collection | DOAJ |
| description | Abstract Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. Results In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. Conclusions By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content. |
| first_indexed | 2024-04-13T13:36:53Z |
| format | Article |
| id | doaj.art-6440344f37f449ecb56698b25b9f35ab |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-04-13T13:36:53Z |
| publishDate | 2017-08-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-6440344f37f449ecb56698b25b9f35ab2022-12-22T02:44:45ZengBMCMicrobial Cell Factories1475-28592017-08-0116111510.1186/s12934-017-0758-xIncreasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthusYing-jie Yang0Ye Wang1Zhi-feng Li2Ya Gong3Peng Zhang4Wen-chao Hu5Duo-hong Sheng6Yue-zhong Li7State Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityState Key Laboratory of Microbial Technology, School of Life Science, Shandong UniversityAbstract Background The CRISPR/Cas9 system is a powerful tool for genome editing, in which the sgRNA binds and guides the Cas9 protein for the sequence-specific cleavage. The protocol is employable in different organisms, but is often limited by cell damage due to the endonuclease activity of the introduced Cas9 and the potential off-target DNA cleavage from incorrect guide by the 20 nt spacer. Results In this study, after resolving some critical limits, we have established an efficient CRISPR/Cas9 system for the deletion of large genome fragments related to the biosynthesis of secondary metabolites in Myxococcus xanthus cells. We revealed that the high expression of a codon-optimized cas9 gene in M. xanthus was cytotoxic, and developed a temporally high expression strategy to reduce the cell damage from high expressions of Cas9. We optimized the deletion protocol by using the tRNA–sgRNA–tRNA chimeric structure to ensure correct sgRNA sequence. We found that, in addition to the position-dependent nucleotide preference, the free energy of a 20 nt spacer was a key factor for the deletion efficiency. Conclusions By using the developed protocol, we achieved the CRISPR/Cas9-induced deletion of large biosynthetic gene clusters for secondary metabolites in M. xanthus DK1622 and its epothilone-producing mutant. The findings and the proposals described in this paper were suggested to be workable in other organisms, for example, other Gram negative bacteria with high GC content.http://link.springer.com/article/10.1186/s12934-017-0758-xCRISPR/Cas9On-target cleavage efficiencySpacer sequenceFree energyDeletion of large genome fragmentsBiosynthetic gene clusters for secondary metabolites |
| spellingShingle | Ying-jie Yang Ye Wang Zhi-feng Li Ya Gong Peng Zhang Wen-chao Hu Duo-hong Sheng Yue-zhong Li Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus Microbial Cell Factories CRISPR/Cas9 On-target cleavage efficiency Spacer sequence Free energy Deletion of large genome fragments Biosynthetic gene clusters for secondary metabolites |
| title | Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus |
| title_full | Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus |
| title_fullStr | Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus |
| title_full_unstemmed | Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus |
| title_short | Increasing on-target cleavage efficiency for CRISPR/Cas9-induced large fragment deletion in Myxococcus xanthus |
| title_sort | increasing on target cleavage efficiency for crispr cas9 induced large fragment deletion in myxococcus xanthus |
| topic | CRISPR/Cas9 On-target cleavage efficiency Spacer sequence Free energy Deletion of large genome fragments Biosynthetic gene clusters for secondary metabolites |
| url | http://link.springer.com/article/10.1186/s12934-017-0758-x |
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