Unmarked gene editing in Clavibacter michiganensis using CRISPR/Cas9 and 5-fluorocytosine counterselection.

Plant pathogenic bacteria in the genus Clavibacter are important quarantine species that cause considerable economic loss worldwide. The development of effective gene editing techniques and additional selectable markers is essential to expedite gene functional analysis in this important Gram-positive genus. The current study details a highly efficient unmarked CRISPR/Cas9-mediated gene editing system in Clavibacter michiganensis (Cm), which couples the expression of cas9 and sgRNA with homology-directed repair templates and the negative selectable marker codA::upp within a single plasmid. Initial experiments indicated that CRISPR/Cas9-mediated transformation could be utilized for both site directed mutagenesis, in which an A to G point mutation was introduced at the 128th nucleotide of the Cm rpsL gene to generate a streptomycin resistant mutant, and complete gene knockout, in which the deletion of the Cm celA or katA genes resulted in transformants that lacked cellulase and catalase activity, respectively. In subsequent experiments the introduction of the codA::upp cassette into the transformation vector facilitated the counterselection of unmarked transformants by incubation in the absence of the selective antibiotic, followed by plating on M9 agar containing 100 μg/ml 5-fluorocytosine (5-FC), in which an unmarked katA mutant lacking the transformation vector was recovered. Compared to conventional homologous recombination, the unmarked CRISPR/Cas9-mediated system was more useful and convenient as it allowed the template plasmid to be reused repeatedly to facilitate the editing of multiple genes, which constitutes a major advancement that could revolutionize research into Cm and other Clavibacter species.