Highly efficient base editing in Staphylococcus aureus using an engineered CRISPR RNA-guided cytidine deaminase

Fig. 1. Comparison between the CRISPR/Cas9-mediated genome editing method and the “cytidine base editor”-mediated genome editing method. (A) Detailed mechanism of the CRISPR/Cas9-mediated genome editing method. (B) The deamination reaction catalyzed by the cytosine deaminase. (C) Detailed mechanism of the “base editor” APOBEC1-nCas9-mediated genome editing method.

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…

Fig. 3. The pnCasSA–BEC system enables highly efficient C → T conversion in the clinically isolated S. aureus strains. (A and B) Q179 of the agrA gene (A) and Q35 of the cntA gene (B) of the S. aureus ST239 (770) strain were successfully mutated to stop codons with 100% efficiency by using the pnCasSA system. ck: the wild-type strain of ST239 (770). (C and D) Q179 of the agrA gene (C) and Q35 of the cntA gene (D) of the S. aureus ST398 (1059) strain were successfully mutated to stop codons with 100% efficiency by using the pnCasSA system. ck: the wild-type strain of ST398 (1059).

Fig. 4. Predicted editable sites of the pnCasSA–BEC system across the entire genome of S. aureus. (A) Proportion of genes containing predicted editable sites of the pnCasSA–BEC system. PAM: PAM sites localized inside genes; mutation: genes containing predicted editable C → T/G → A conversion sites; mutation stop: C → T/G → A conversions generate premature stop codons in genes. (B) Cumulative curve of the distribution of the generated premature stop codons inside gene bodies. (C) Circos plot showing the distribution of genes that could be edited by the pnCasSA–BEC system in the genome of MRSA252. Concentric circles from the inside to the outside: (13) CG content; (12) genes with stop mutation sites; (11) genes with C–T mutation sites; (10) genes with PAM sites; (9) CG offsets ((G – C)/(G + C)); (8–5) genes with stop mutation sites in 0–20%, 20–50%, 50–80%, and 80–100% of the gene body; (4) gene annotations; (3–2) distribution of the genes in forward and reverse strands of the genome; (1) the distribution of the genes in the entire genome.