Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae

The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of . One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of . Here we exploited this system further under challenging conditions: by targeting the AMR gene located on a high-copy plasmid (i.e., 100-300 copies/cell) and by directly tackling -positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of harboring on the plasmid pSB1A2, the plasmid number and, accordingly, the gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of , plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of infected with CRISPR-Cas9-treated , as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of and to a lesser extent in , both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.