CRISPR-Cas9 Based Engineering of Actinomycetal Genomes

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


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.

Keywords: CRISPR-Cas9; CRISPRi; DNA repair; actinomycetes; genome engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA Breaks, Double-Stranded
  • DNA, Bacterial / genetics
  • Gene Expression Regulation, Bacterial*
  • Genetic Engineering*
  • Genome, Bacterial*
  • Molecular Sequence Data
  • Multigene Family
  • Sequence Analysis, DNA
  • Streptomyces coelicolor / genetics*
  • Streptomyces coelicolor / metabolism


  • DNA, Bacterial

Associated data

  • GENBANK/KR011748
  • GENBANK/KR011749