title: High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system.
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.
authors.affiliation: †Department of Chemical and Biomolecular Engineering, ‡Institute for Genomic Biology, §Departments of Chemistry, Biochemistry and Bioengineering, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Cobb
authors.affiliation: †Department of Chemical and Biomolecular Engineering, ‡Institute for Genomic Biology, §Departments of Chemistry, Biochemistry and Bioengineering, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Yajie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: †Department of Chemical and Biomolecular Engineering, ‡Institute for Genomic Biology, §Departments of Chemistry, Biochemistry and Bioengineering, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D017353:Gene Deletion; D005818:Genetic Engineering; D016681:Genome, Fungal; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
keywords: CRISPR; Cas9; Streptomyces; genome engineering; synthetic guide RNA