Engineered CRISPR-Cas9 for Streptomyces sp. genome editing

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 implemented 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, was 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 demonstrated improved secondary metabolite production using multiplexed genome editing with multiple single guide RNA libraries in several Streptomyces strains. Cas9-BD was also used to capture large BGCs using a newly developed in vivo cloning method. The modified CRISPR-Cas9 system was successfully applied to many Streptomyces sp., providing versatile and efficient genome editing tools for strain engineering of actinomycetes with high GC content genome.