Staphylococcus aureus plasmids and schematic representation of genome-editing methods using nCas9-deaminase fusion enzymes. (A) Map of the S. aureus pnCasSA-BEC plasmid used for the expression of the APOBEC1–nCas9 fusion enzyme, with relevant features indicated. (B) Map of the S. aureus pABE plasmid used for the expression of the ABE7.10–nCas9 fusion enzyme, with relevant features indicated. (C) Schematic representation of the process leading to a C-to-T conversion using the APOBEC1–nCas9 fusion enzyme. The APOBEC1–nCas9 fusion enzyme bound to an appropriately designed synthetic guide RNA (sgRNA) will bind to the chromosome around a protospacer-adjacent motif (PAM) site. The nCas9 enzyme will introduce a nick in the bottom DNA strand, whereas the APOBEC1 deaminase will convert an appropriately spaced cytosine (C) in the top strand to a uracil (U). This will lead to a C-to-T base conversion during DNA replication. (D) Schematic representation of the process leading to an A-to-G conversion using the ABE7.10–nCas9 fusion enzyme. The ABE7.10–nCas9 enzyme bound to an appropriately designed sgRNA will bind to the chromosome around a PAM site. The nCas9 enzyme will introduce a nick in the bottom DNA strand and the ABE7.10 deaminase will then convert an appropriately spaced adenine (A) in the top strand to a hypoxanthine (I). This will result in an A-to-G conversion during DNA replication. Plasmid maps shown in panels A and B were generated using SnapGene.