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ACS Synth Biol. 2015 Sep 18;4(9):1020-9. doi: 10.1021/acssynbio.5b00038. Epub 2015 Apr 7.

CRISPR-Cas9 Based Engineering of Actinomycetal Genomes.

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The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Alle 6, Hørsholm 2970, Denmark.
Department of Bioengineering, University of California, San Diego , La Jolla, California 92093, United States.
Chinese Academy of Sciences, Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology , Beijing 100190, China.
Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST) , Daejeon 305-701, Republic of Korea.


Bacteria of the order Actinomycetales are one of the most important sources of pharmacologically active and industrially relevant secondary metabolites. Unfortunately, many of them are still recalcitrant to genetic manipulation, which is a bottleneck for systematic metabolic engineering. To facilitate the genetic manipulation of actinomycetes, we developed a highly efficient CRISPR-Cas9 system to delete gene(s) or gene cluster(s), implement precise gene replacements, and reversibly control gene expression in actinomycetes. We demonstrate our system by targeting two genes, actIORF1 (SCO5087) and actVB (SCO5092), from the actinorhodin biosynthetic gene cluster in Streptomyces coelicolor A3(2). Our CRISPR-Cas9 system successfully inactivated the targeted genes. When no templates for homology-directed repair (HDR) were present, the site-specific DNA double-strand breaks (DSBs) introduced by Cas9 were repaired through the error-prone nonhomologous end joining (NHEJ) pathway, resulting in a library of deletions with variable sizes around the targeted sequence. If templates for HDR were provided at the same time, precise deletions of the targeted gene were observed with near 100% frequency. Moreover, we developed a system to efficiently and reversibly control expression of target genes, deemed CRISPRi, based on a catalytically dead variant of Cas9 (dCas9). The CRISPR-Cas9 based system described here comprises a powerful and broadly applicable set of tools to manipulate actinomycetal genomes.


CRISPR-Cas9; CRISPRi; DNA repair; actinomycetes; genome engineering

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