Recombineering in Staphylococcus aureus.

Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.