AbstractBackgroundRhodobacter sphaeroidesis a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-β-hydroxybutyrate (PHB), has been an important metabolic engineering target forR. sphaeroides. However, the lack of efficient markerless genome editing tools forR. sphaeroidesis a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system ofStreptococcus pyogenes(SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not forR. sphaeroides.ResultsHere we describe the development of a highly efficient SpCas9-based genomic DNA targeting system forR. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp)genefrom the genome ofR. sphaeroides,as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genesphaBandphbBin the genome ofR. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of theR. sphaeroidesΔphbBstrains under the same conditions is only slightly affected.ConclusionsIn this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism ofR. sphaeroidesfor the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway ofR. sphaeroides.We anticipate that the presented work will accelerate molecular research withR. sphaeroides.