Gene editing with CRISPR-CAS system in multidrug resistant klebsiella pneumoniae isolates isolated from clinical specimens

In this study, it was aimed to edit the gene in multidrug resistant Klebsiella pneumoniae isolates isolated from clinical samples at Department of Clinical Microbiology, Istanbul Faculty of Medicine, Istanbul University. Five multidrug resistant K. pneumoniae strains isolated between June and September 2020 were included in the study. The minimum inhibitory concentration (MIC) values for imipenem, meroperem and ertapenem in these isolates were determined by broth microdilution method according to EUCAST recommendations. The presence of blaOXA-48, blaNDM-1 and blaCTX-M-15 genes of isolates was investigated by classical polymerase chain reaction (PCR) method. A dual plasmid (CRISPR-Cas9 and CRISPR-assisted cytidine deaminase) was designed in the CRISPR-Cas system. The susceptibility of the antibiotics to be used in the selection of plasmids for transformation to the isolates was investigated. Targeted gene regions (AmpR (Pro), AraBAD, ApmR, Cas9 and Cas9 (D10A)-APOBEC1) was amplified by PCR method. Cloning was done by ligation and TEDA (T5 exonuclease DNA assembly) method. The MIC values of the isolates between 4-128 µg/mL for imipenem, 8-64 µg/mL for meropenem, and 8-256 µg/mL for ertapenem. All isolates were resistant to ertapenem, meropenem and imipenem. The blaOXA-48-like and blaCTX-M-15 genes were detected in all isolates (n=5), and blaNDM-1 gene was detected in one isolate (20%). According to the results of the sequence analysis of the gene regions, gRNA sequences were created. It was found sensitive only to apramycin (50 µg/mL) among the antibiotics to be used in selection in transformation. Cas9 and APOBEC regions could not be cloned into the backbone vector designed for use in the CRISPR-Cas system, but the pSGKP-AmpR(Pro)-AprR plasmid was successfully created. It is the first study in our country in which gene editing was performed with the CRISPR-Cas system in bacteria resistant to antibiotics. In conclusion, it is predicted that gene editing can be done by using the CRISPR-Cas system in K. pneumoniae isolates that are multidrug resistant to antibiotics, this is the first study in our country, and it can be an alternative solution to antibiotic treatments in resistant bacteria, supported by more and further studies.