Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen responsible for a variety of infectious diseases. This pathogen possesses defense mechanisms that allow it to resist multiple β-lactam antibiotics. Previous research has shown that the serine-threonine kinase, Stk1, is a master regulator of antibiotic resistance in S. aureus. Our lab has successfully identified putative small molecule inhibitors of Stk1 that potentiate β-lactam antibiotics. We set out to create stk1 deletion mutants that can be used as genetic tools for studying Stk1 function. To accomplish this, we used the CRISPR/Cas9 system, which has previously been used to edit two clinically relevant strains of MRSA. The CRISPR plasmids purified from E. coli were first transformed into an intermediate laboratory strain, RN4220, that lacks the restriction barriers other S. aureus strains possess. We optimized our methods for preparing the RN4220 cells and CRISPR plasmids prior to electroporation. Purified plasmid from RN4220 was then electroporated into our chosen clinically relevant strains of MRSA. PCR and DNA sequencing were used to verify the deletion of stk1. This genetic editing tool will also allow for the deletion of other genes that may contribute to the antibiotic resistance of MRSA and allow for the creation of point mutations in the S. aureus genome. We can also gain a better understanding of how Stk1 functions in the regulation of antibiotic resistance in a variety clinically relevant strains of MRSA.

CRISPR/Cas9 as a genetic tool for studying MRSA antibiotic resistance

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