caa a22 4500 605502676 CHVBK 20210128100607.0 cr unu---uuuuu 210128e20151201xx s 000 0 eng 10.1007/s00253-015-6931-4 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6931-4 Highly efficient editing of the actinorhodin polyketide chain length factor gene in Streptomyces coelicolor M145 using CRISPR/Cas9-CodA(sm) combined system [Elektronische Daten] [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. Springer-Verlag Berlin Heidelberg, 2015 CRISPR/Cas9 nationallicence CodA nationallicence Actinorhodin biosynthesis nationallicence ActI-ORF2 nationallicence Gene editing nationallicence Zeng Hu Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Wen Shishi Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Xu Wei Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut He Zhaoren Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Zhai Guifa Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Liu Yunkun Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Deng Zixin Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Sun Yuhui Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10575-10585 0175-7598 99:24<10575 2015 99 253 https://doi.org/10.1007/s00253-015-6931-4 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6931-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Zeng Hu Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Wen Shishi Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Xu Wei Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- He Zhaoren Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Zhai Guifa Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Liu Yunkun Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Deng Zixin Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 700 1- Sun Yuhui Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University), Ministry of Education and Wuhan University School of Pharmaceutical Sciences, 185 East Lake Road, 430071, Wuhan, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/24(2015-12-01), 10575-10585 0175-7598 99:24<10575 2015 99 253