CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous <i>Rhodococcus</i>

Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70–80% and 75–85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.