Abstract
ABSTRACTStaphylococcus aureus is 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. aureus using 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. aureus genome. We found that EF2132 can 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. aureus strains 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. aureus without 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. aureus genome 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. aureus which will enable the high-throughput functional assessment of genome variation and gene function across multiple strain backgrounds.
Highlights
Staphylococcus aureus is 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
We evaluated six different recombinases exogenously expressed from cassette-based shuttle vector pCN50 [36]: bet, the recombinase gene utilized in the E. coli Red recombineering system [25]; EF2132 and orfC, derived from Enterococcus faecalis and Legionella pneumophilia, respectively, both being genes that have been previously shown to have cross-species activity in E. coli [25]; gp20, a recombinase originating from S. aureus with weak activity in E. coli [25]; and two putative S. aureus recombinases which we identified on the basis of protein homology to these known recombinases
Each expression construct was separately introduced into S. aureus type strain ATCC 29213 (Rosenbach [37]), electrocompetent cells were prepared, and mutagenic oligonucleotide was subsequently introduced by electroporation
Summary
Staphylococcus aureus is 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. 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. aureus genome and for eliminating cells that lack the engineered mutation This method allows efficient, precise, and highthroughput genetic engineering of S. aureus strains and will facilitate studies seeking to address a variety of issues about the function of particular genes and specific mutations. Most established methods for modifying S. aureus genomes rely on rare homologous recombination events with large donor fragments encoding the desired change, with antibiotic-mediated selection for successful allelic exchange [17,18,19] Useful, these techniques are relatively inefficient and introduce exogenous genetic material into the host genome along with the targeted mutation [18, 20]. All available genome editing strategies for S. aureus remain laborious and involve the individual cloning of each ~1-kb-to-2-kb homologous repair template, with accompanying protocol optimization [17, 18, 20,21,22, 24]; this strategy is especially difficult to implement for engineering gene deletions, which must be manufactured using splicing by overhang extension (SOE) PCR [15, 23]
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