Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production
Abstract The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipula...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-56278-y |
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| author | Duck Gyun Kim Boncheol Gu Yujin Cha Jeonghan Ha Yongjae Lee Gahyeon Kim Byung-Kwan Cho Min-Kyu Oh |
| author_facet | Duck Gyun Kim Boncheol Gu Yujin Cha Jeonghan Ha Yongjae Lee Gahyeon Kim Byung-Kwan Cho Min-Kyu Oh |
| author_sort | Duck Gyun Kim |
| collection | DOAJ |
| description | Abstract The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome. |
| format | Article |
| id | doaj-art-4834d17b26db4ecda07483cfd789f301 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-4834d17b26db4ecda07483cfd789f3012025-01-26T12:41:45ZengNature PortfolioNature Communications2041-17232025-01-0116111510.1038/s41467-025-56278-yEngineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite productionDuck Gyun Kim0Boncheol Gu1Yujin Cha2Jeonghan Ha3Yongjae Lee4Gahyeon Kim5Byung-Kwan Cho6Min-Kyu Oh7Department of Chemical & Biological Engineering, Korea UniversityDepartment of Chemical & Biological Engineering, Korea UniversityDepartment of Chemical & Biological Engineering, Korea UniversityDepartment of Chemical & Biological Engineering, Korea UniversityDepartment of Biological Sciences, Korea Advanced Institute of Science and TechnologyDepartment of Biological Sciences, Korea Advanced Institute of Science and TechnologyDepartment of Biological Sciences, Korea Advanced Institute of Science and TechnologyDepartment of Chemical & Biological Engineering, Korea UniversityAbstract The CRISPR-Cas9 system has frequently been used for genome editing in Streptomyces; however, cytotoxicity, caused by off-target cleavage, limits its application. In this study, we implement innovative modification to Cas9, strategically addressing challenges encountered during gene manipulation using Cas9 within strains possessing high GC content genome. The Cas9-BD, a modified Cas9 with the addition of polyaspartate to its N- and C-termini, is developed with decreased off-target binding and cytotoxicity compared with wild-type Cas9. Cas9-BD and similarly modified dCas9-BD have been successfully employed for simultaneous biosynthetic gene cluster (BGC) refactoring, multiple BGC deletions, or multiplexed gene expression modulations in Streptomyces. We also demonstrate improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD is also used to capture large BGCs using a developed in vivo cloning method. The modified CRISPR-Cas9 system is successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.https://doi.org/10.1038/s41467-025-56278-y |
| spellingShingle | Duck Gyun Kim Boncheol Gu Yujin Cha Jeonghan Ha Yongjae Lee Gahyeon Kim Byung-Kwan Cho Min-Kyu Oh Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production Nature Communications |
| title | Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production |
| title_full | Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production |
| title_fullStr | Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production |
| title_full_unstemmed | Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production |
| title_short | Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production |
| title_sort | engineered crispr cas9 for streptomyces sp genome editing to improve specialized metabolite production |
| url | https://doi.org/10.1038/s41467-025-56278-y |
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