CRISPR/Cas9-Mediated Promoter Engineering in Saccharopolyspora erythraea

In situ promoter engineering is an effective way to alter target gene expression without introducing excess DNA sequences. Recently, the CRISPR/Cas9 technologies have been proved to be efficient tools for genome editing in actinomycetes, making it easier and more efficient to perform gene insertion and substitution in actinomycetes in a scarless manner. In this chapter, we describe a routine protocol for CRISPR/Cas9-mediated promoter engineering in Saccharopolyspora erythraea NRRL 23338, which is the wild-type producer of erythromycin. This protocol can be adapted to CRISPR/Cas9-mediated gene editing, not limited to promoter engineering, in other actinomycetes, with modifications.