Developing a Streptomyces-based cell factory is key for the production of new or known biologically active compounds. With gradual deletion of large genomic regions one can develop an efficient platform for the heterologous expression of secondary metabolites. Gene manipulations of Streptomyces using classical approaches are demanding and time-consuming. In this work we used CRISPR-Cas9 to achieve a 145 kb genomic deletion in Streptomyces rimosus. With the use of bioinformatics we chose a target region with accompaning homologies and guide RNAs. We then used overlap PCR and SLiCE cloning to construct a plazmid for CRISPR-Cas9 driven deletion. The plasmid was introduced into S. rimosus with conjugation, then positive clones were tested for the deletion using PCR and sequencing. We have identified several isolates with correctly deleted DNA fragment of 145 kb in size.
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