Efficient Genome Editing in Most by Using the Restriction-Modification System Silent CRISPR-Cas9 Toolkit.

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 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 . Here, we constructed a restriction-modification (RM) system silent CRISPR-Cas9 toolkit that synonymously eliminated the type I RM targets of 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 (10-10 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 species including clinical isolates of unknown genetic background. The RM-silent CRISPR-Cas9 toolkits could expedite the process of mutant construction in most strains, and this approach could be applied to the design of other genetic toolkit plasmids for utilization in a wider range of strains.