Engineered CRISPR-Cas9 for Streptomyces sp. genome editing to improve specialized metabolite production

Duck Gyun Kim, Boncheol Gu, Yujin Cha, Jeonghan Ha, Yongjae Lee, Gahyeon Kim, Byung-Kwan Cho and Min-Kyu Oh ()
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Duck Gyun Kim: Korea University
Boncheol Gu: Korea University
Yujin Cha: Korea University
Jeonghan Ha: Korea University
Yongjae Lee: Korea Advanced Institute of Science and Technology
Gahyeon Kim: Korea Advanced Institute of Science and Technology
Byung-Kwan Cho: Korea Advanced Institute of Science and Technology
Min-Kyu Oh: Korea University

Nature Communications, 2025, vol. 16, issue 1, 1-15

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-56278-y

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