CRISPR-Cas based plasmid design for multidrug-resistant Klebsiella pneumoniae isolates.

Antimicrobial resistance is a major global health concern that requires innovative therapeutic strategies. This study aimed to address this challenge by designing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (CRISPR-Cas)-based plasmid systems for potential genome editing applications in multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined according to European Committee on Antimicrobial Susceptibility Testing guidelines. All isolates (n = 5) were resistant, with MIC ranges of 4-128 μg/ml for imipenem, 8-64 μg/ml for meropenem, and 8-256 μg/ml for ertapenem. Resistance gene analysis revealed blaOXA-48-like and blaCTX-M-15 in all isolates, while blaNDM-1 was detected in one isolate. Two CRISPR-based plasmid systems, CRISPR-Cas9 and CRISPR-assisted cytidine deaminase, were designed. Target genes were amplified by polymerase chain reaction, and guide RNA (gRNA) sequences were designed from selected regions. Apramycin (50 μg/ml) was identified as a suitable selection marker. The pSGKP-AmpR(Pro)-ApmR plasmid was successfully constructed, whereas Cas9 and APOBEC constructs could not be cloned. Overall, this study highlights technical challenges in developing CRISPR-based tools for MDR K. pneumoniae and emphasizes the need for isolate-specific plasmid design and gRNA optimization.