Developing a Base Editing System for Marine Roseobacter Clade Bacteria

The Roseobacter clade bacteria are of great significance in marine ecology and biogeochemical cycles, and they are potential microbial chassis for marine synthetic biology due to their versatile metabolic capabilities. Here, we adapted a CRISPR-Cas-based system, base editing, with the combination of nuclease-deactivated Cas9 and deaminase for Roseobacter clade bacteria. Taking the model roseobacter Roseovarius nubinhibens as an example, we achieved precise and efficient genome editing at single-nucleotide resolution without generating double-strand breaks or requesting donor DNAs. Since R. nubinhibens can metabolize aromatic compounds, we interrogated the key genes in the β-ketoadipate pathway with our base editing system via the introduction of premature STOP codons. The essentiality of these genes was demonstrated, and for the first time, we determined PcaQ as a transcription activator experimentally. This is the first report of CRISPR-Cas-based genome editing in the entire clade of Roseobacter bacteria. We believe that our work provides a paradigm for interrogating marine ecology and biogeochemistry with direct genotype-and-phenotype linkages and potentially opens a new avenue for the synthetic biology of marine Roseobacter bacteria.