Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system
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The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing toolkits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counterselection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes.
KeywordsCRISPR/Cas9 CodA Actinorhodin biosynthesis ActI-ORF2 Gene editing
This research was supported by grants from National Basic Research Program of China (973 Program), Research Fund for the Doctoral Program of Higher Education of China, and Program for Collaborative Innovative in Wuhan University School of Medicine. We thank Dr. Ryszard Brzezinski at Universite´ de Sherbrooke in Canada for the gift of pMG303M containing codA(sm). We are grateful to associate Prof. Changming Zhao for his help in LC-ESI-HRMS detection of actinorhodin and Dr. Tobias Kieser for his critical reading of the manuscript.
This article does not contain any studies with human participants or animals performed by any of the authors.
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