Efficient Genome Editing in Most <i>Staphylococcus aureus</i> by Using the Restriction-Modification System Silent CRISPR-Cas9 Toolkit

Staphylococcus aureus is a clinically important pathogen that threatens human health due to its strong pathogenicity and drug resistance, leading to meningitis, endocarditis, and skin and soft tissue infections. Genetic manipulation in S. aureus is a powerful approach for characterizing the molecular mechanisms of bacterial drug resistance, pathogenicity, and virulence. However, a strong restriction barrier presents a major obstacle to the extensive utilization of genetic manipulation tools in clinical isolates of S. aureus. Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of S. aureus from plasmids, downsized plasmids using minicircle technology, and combined with a plasmid artificial modification (PAM) method to circumvent the type II RM system. The RM-silent CRISPR-Cas9 toolkit enables a significant improvement in transformation (105–106 transformants per microgram plasmid in strains we tested) and high-success efficiency editing for gene deletion (knockout strain obtained in one-round electroporation) in a wide range of S. aureus species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most S. aureus strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of S. aureus strains