Efficient editing DNA regions with high sequence identity in actinomycetal genomes by a CRISPR-Cas9 system

Actinobacteria able to produce varieties of bioactive natural products have been long appreciated by the field of drug discovery and development. Recently, a few of CRISPR/Cas9 systems bearing different types of replicons (pSG5 and pIJ101) were developed to efficiently edit their genomes. Despite wide application in gene editing, their utility in editing challenging DNA regions e.g. high sequence identity has not been compared. In this study, we confirmed that the widely used temperature-sensitive pSG5 replicon is indeed not suitable for editing modular polyketide synthase (PKS) genes due to causing unpredicted gene recombination. This problem can be addressed by replacing the pSG5 with the segregationally unstable pIJ101 replicon. By introducing a counter-selection marker CodA, convenient cloning sites in the single guide RNAs (sgRNAs) and homologous template scaffolds, we developed a new CRISPR-Cas9 system pMWCas9. This system was successfully used to delete/replace erythromycin PKS and other biosynthetic genes in Saccharopolyspora erythraea and Streptomyces sp. AL2110. By swapping the promoters of antB and antC with ermE and kasOp, we achieved a deacyl-antimycin hyper producer which produces a 9-fold higher yield than the original Streptomyces sp. AL2110 strain. Our results provide a robust and useful Cas9 tool for genetic studies in Actinobacteria.