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ABSTRACTStaphylococcus aureusis an important human pathogen, but studies of the organism have suffered from the lack of a robust tool set for its genetic and genomic manipulation. Here we report the development of a system for the facile and high-throughput genomic engineering of S. aureususing single-stranded DNA (ssDNA) oligonucleotide recombineering coupled with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated counterselection. We identify recombinase EF2132, derived from Enterococcus faecalis, as being capable of integrating single-stranded DNA oligonucleotides into the S. aureusgenome. We found that EF2132can readily mediate recombineering across multiple characterized strains (3 of 3 tested) and primary clinical isolates (6 of 6 tested), typically yielding thousands of recombinants per transformation. Surprisingly, we also found that some S. aureusstrains are naturally recombinogenic at measurable frequencies when oligonucleotides are introduced by electroporation, even without exogenous recombinase expression. We construct a temperature-sensitive, two-vector system which enables conditional recombineering and CRISPR/Cas9-mediated counterselection in S. aureuswithout permanently introducing exogenous genetic material or unintended genetic lesions. We demonstrate the ability of this system to efficiently and precisely engineer point mutations and large single-gene deletions in the S. aureusgenome and to yield highly enriched populations of engineered recombinants even in the absence of an externally selectable phenotype. By virtue of utilizing inexpensive, commercially synthesized synthetic DNA oligonucleotides as substrates for recombineering and counterselection, this system provides a scalable, versatile, precise, inexpensive, and generally useful tool for producing isogenic strains in S. aureuswhich will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.IMPORTANCEEngineering genetic changes in bacteria is critical to understanding the function of particular genes or mutations but is currently a laborious and technically challenging process to perform for the important human pathogen Staphylococcus aureus. In an effort to develop methods which are rapid, easy, scalable, versatile, and inexpensive, here we describe a system for incorporating synthetic, mutagenic DNA molecules into the S. aureusgenome and for eliminating cells that lack the engineered mutation. This method allows efficient, precise, and high-throughput genetic engineering of S. aureusstrains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations.