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. 2021 Feb 28;49(6):3584–3598. doi: 10.1093/nar/gkab126

Figure 4.

Figure 4.

TAP re-sensitises pOXA48-carrying recipient cells and impedes resistance dissemination. (A) Diagram of the TAP-mediated anti-resistance strategy. TAP-Cas9-OXA48 targeting the blaOXA48 promoter is transferred from a donor to an ampicillin resistant recipient cells carrying the pOXA-48a plasmid. Acquisition of the TAP-Cas9-OXA48 induces double-strand-breaks (DSBs) into the plasmid, while the TAP-dCas9-OXA48 inhibits blaOXA48 gene expression. Both TAPs sensitize the transconjugant cells to ampicillin. (B) Histograms showing reduction of ampicillin resistance in transconjugants cells after acquisition of the TAPkn-Cas9-OXA48, TAPkn-dCas9-OXA48 and TAPkn-Cas9-OXA48-PemI. Percentages of transconjugants (ratio T/R + T) are indicated. (C) Histograms showing the frequency of donor cells acquiring ampicillin resistance through transfer of pOXA-48a from the recipients (as depicted in the above diagram). (D) Histograms show the frequency of ampicillin-resistance acquisition through pOXA-48a transfer into R#2 plasmid-free wt recipient that have received the TAPs (TAP-transc.#2) (as depicted in the above diagram). Mean and SD are calculated from at least three independent experiments. Donors TAP-dCas9-nsp (LY1524), TAP-Cas9-nsp (LY1369), TAP-dCas9-OXA48 (LY1523), TAP-Cas9-OXA48 (LY1522) or TAP-PemI-Cas9-OXA48 (LY1549); Recipients R#1 wt/pOXA-48a (LY1507) and R#2 wt (LY945). (B–D) Two-tailed unpaired t-tests were performed. ns = non-significant P-value >0.05; *P-value <0.05, **P-value <0.0021; ****P-value <0.0001.