Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system

Hu Zeng, Shishi Wen, Wei Xu, Zhaoren He, Guifa Zhai, Yunkun Liu, Zixin Deng, Yuhui Sun

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.