title: Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system.
abstract: 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.
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Hu
authors: H
authors: Zeng
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Shishi
authors: S
authors: Wen
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Wei
authors: W
authors: Xu
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Zhaoren
authors: Z
authors: He
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Guifa
authors: G
authors: Zhai
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Yunkun
authors: Y
authors: Liu
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China.
authors: Zixin
authors: Z
authors: Deng
authors: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, Wuhan, 430071, People's Republic of China. yhsun@whu.edu.cn.
authors: Yuhui
authors: Y
authors: Sun
mesh_terms: D000880:Anthraquinones / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D060847:Metabolic Engineering; D009154:Mutation; D061065:Polyketides / Q000378:metabolism*; D011995:Recombination, Genetic; D012641:Selection, Genetic; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism*; D000095622:Benzoisochromanequinones
chemical_list: D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D061065:Polyketides; C013390:actinorhodin; D000095622:Benzoisochromanequinones
keywords: ActI-ORF2; Actinorhodin biosynthesis; CRISPR/Cas9; CodA; Gene editing