A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus, even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, RrRecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40–80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 103-fold higher than that of endogenous pathways. Finally, by incorporating RrRecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus, with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.