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.

Keywords: CRISPR; Cas9; Streptomyces; genome engineering; synthetic guide RNA.

Figure 1

pCRISPomyces plasmids for targeted genome editing in Streptomyces species. Notable features include a codon-optimized cas9 from Streptococcus pyogenes, a BbsI-flanked lacZ cassette for Golden Gate assembly of spacer sequences, an XbaI site for addition of editing templates, and a temperature-sensitive pSG5 origin.

Figure 2

(a) Strategy for deletion of the 31kb red cluster. Two sgRNA transcripts guide Cas9 to introduce DSBs at both ends of the cluster, while a codelivered editing template bridges the gap via homologous recombination. (b) PCR evaluation of red cluster deletion from four exconjugants (DF1–DF4) with wild type control (wt). (c) Phenotypic evaluation of strains DF1–DF4 with wild type control.