Fig. 2. The engineered base-editing system enables highly efficient C → T conversion in the S. aureus RN4220 strain. (A) Map of the base-editing plasmid pnCasSA–BEC. APOBEC1-nCas9, a fusion protein composed of a deaminase APOBEC1 at the N terminus and a Cas9 nickase Cas9D10A at the C terminus; rpsL promoter, an S. aureus strong promoter used to drive the expression of APOBEC1-nCas9; BsaI sites, Golden Gate assembly of spacers; cap 1A promoter, the sgRNA expression promoter; ColE1, an E. coli replication origin. KanR, the antibiotic marker kanamycin used in E. coli; Cm, the chloramphenicol-resistance marker used in S. aureus; repF, an S. aureus temperature-sensitive origin for plasmid curing after editing. (B) The potential editable sites in the genome. The prediction of the potential editable sites is based on the mammalian “base editor” BE3.19 (C and D) The pnCasSA–BEC system enables highly efficient base editing in the RN4220 strain. Both the mutation efficiencies of agrA Q179 to stop codon (C) and cntA Q35 to stop codon (D) were 100%. The PAM sites are colored blue. The spacers are colored green. The mutation sites are colored red. A representative sequencing chromatogram for each mutation is shown.