Abstract Background Genetic manipulation of Staphylococcus aureus (both methicillin sensitive S. aureus, MSSA, and methicillin resistant S. aureus, MRSA) poses a technical challenge due to poor transformation efficiency, limited endogenous DNA repair activity, lineage-specific methylation patterns and intrinsic resistance to common selectable markers. Methods To address transformation efficiency we have optimized electrocompetent cell preparation and electroporation protocols for staphylococci. Further, we have improved a CRISPR counterselection platform that delivers a heterologous ssDNA recombinase and an inducible Cas9 endonuclease. When used for recombineering and counterselection, this strategy allows minimization of the number of elements necessary to transform in a single electroporation event. The Cas9 delivery platform has been modified to include a range of selectable markers including resistance to apramycin, erythromycin, kanamycin, nourseothricin, spectinomycin or trimethoprim. Results Overall electroporation efficiency increased by multiple orders of magnitude (> 100×) using the optimized cell preparation protocol. The CRISPR delivery platform can be stably maintained in a repressed state for multiple generations and induced with anhydrotetracycline. We have introduced targeted mutations in multiple loci using this system with an average turnaround time of 12 days. Conclusion This improved dual-plasmid CRISPR platform is robust and allows the investigator to rapidly and specifically alter the genomes of staphylococci. These tools will facilitate the study of how specific genetic polymorphisms contribute to various phenotypes in S. aureus, including the virulence of MRSA. Disclosures All Authors: No reported disclosures