Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene bla NDM-1 , with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes bla NDM-1 and bla SHV-12 , with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the bla NDM-1 promoter region can silence the entire bla NDM-1 -ble MBL -trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene bla KPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.