The use of a CRISPR-Cas9 system to protect probiotic strains from transferrable drug resistance genes

Abstract: The discovery of antibiotics have revolutionized modern medicine, facilitating the treatment of a variety of bacterial infections, enabled surgeries otherwise impossible to perform and increased life expectancy in all countries. However, the rapid development of resistance among microorganisms and the increasing numbers of clinical outbreaks caused by multiresistant bacteria have accelerated the need for new alternatives to antibiotics. Probiotic bacteria armed with defense systems have been studied as potential substitutes of antibiotics. These probiotic competitors can still contribute to the spread of resistance genes among microorganisms through horizontal gene transfer. The aim of this study was to investigate whether constructed CRISPR-Cas9 systems have the potential to protect probiotic bacteria against horizontal transfer of antibiotic resistance genes. Transformation, transduction and conjugation assays in strains carrying or not carrying a plasmid-bourne CRISPR-Cas9 system were performed in order to compare the frequencies of transfer of the most common resistance genes. The transformation and transduction assays demonstrated that the constructed CRISPR-Cas9 system entails a decrease in efficiency of transfer for targeted resistance genes. Moreover, it can be concluded that potentially increasing Cas9 levels by reducing its degradation results in increased prevention of horizontal gene transfer through transformation and transduction. Finally, we state that the CRISPRCas9 system does not result in protection against antibiotic resistance genes entering the cells through conjugation.