High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System

Ryan E. Cobb, Yajie Wang, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces.

Original languageEnglish (US)
Pages (from-to)723-728
Number of pages6
JournalACS synthetic biology
Volume4
Issue number6
DOIs
StatePublished - Jun 19 2015

Keywords

  • CRISPR
  • Cas9
  • Streptomyces
  • genome engineering
  • synthetic guide RNA

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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