Endogenous Staphylococcus aureus CRISPR-cas system limits phage proliferation and efficiently excises from the genome as part of the SCCmec cassette

CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR- cas , however in the human pathogen Staphylococcus aureus , CRISPR- cas loci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR- cas in genomes of methicillin resistant Staphylococcus aureus (MRSA) isolated in Denmark. Only 2.9 % of the strains carried CRISPR- cas systems, but for strains of sequence type ST630 over half were positive. All CRISPR- cas loci were type III-A and located within the staphylococcal chromosomal cassette (SCC mec ) type V(5C2&5) conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-positive strains and almost identical SCC mec cassettes, CRISPR arrays and cas genes, are present in staphylococcal species other than aureus , suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCC mec cassette containing CRISPR- cas excises from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage (phage) virus philPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or bypassed by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system in S. aureus is active against targeted phages, albeit with low efficacy. This suggests native S. aureus CRISPR-Cas offers only partial immunity, and in nature may work in tandem with other defense systems. Importance CRISPR-Cas is an adaptive immune system enabling bacteria and archaea to protect themselves against mobile genetic elements such as phages. In strains of Staphylococcus aureus , CRISPR- cas is rare, but when present, it is located within the SCC mec element encoding resistance to methicillin and other β-lactam antibiotics. We show that the entire module is excisable, with almost identical versions found in different species of non-aureus staphylococci suggesting that the system only rarely acquires new spacers in S. aureus . Additionally, we show that in its endogenous form, the S. aureus CRISPR-Cas is active but inefficient against lytic phages, with phages being able to form escape mutants or overload the system. This leads us to propose that CRISPR-Cas in S. aureus offers only partial immunity in native systems, and so may work together with other defense systems to prevent phage-mediated killing. ### Competing Interest Statement The authors have declared no competing interest.