The Application Of CRISPR/Cas9 Technology In The Investigation Of The Mechanism Of Action Of The Fluoroquinolone C-7 Substituent

The extensive use of fluoroquinolones resulted in many drug-resistant mutant strains, thus, to explore the strains' resistance mechanisms and develop better antiseptic became the most important work in the whole medical industry. Now, the enhancement of the performance of fluoroquinolones mainly by modifying the substituents on the nucleus C, Wherein, the C-7 position substituent is variable, and has good prospects for the use in the development of drug, so in-depth study of C-7 substituent mechanism in fluoroquinolones sterilization process has great significance. In addition, according to preliminary study, we found that the end of fluoroquinolone C-7 ring fell at gyrB466 exactly, but because we had not received 466 single mutants yet, we can not reveal the strength of their relationship.CRISPR/Cas9 is an emerging gene editing technology, which is efficient and accurate, and what's more, it plays an important role in the study of gene function. In this paper, we constructed gyrB466 C mutants by using CRISPR/Cas9 coupled ?-red homologous recombination technology, and then performed the susceptibility testing to analyze the relationship between gyrB466 amino acid and fluoroquinolones, the main content and results of the experiment are as follows:We successfully substituted GAA which encoded the E. coli MG1655 gyrB466 amino acid for TGT by building CRISPR/Cas9 gene editing systems and ?-red homologous recombination system, and the replacement efficiency is up to 100%. And subsequently, the ciprofloxacin susceptibility testing were implemented, results showed that: the minimum inhibitory concentration(MIC) of the mutant strain is four times of the wild strain, further the pharmacokinetic experiments showed that the absolute death concentration(LD100) is 0.2?g/ml which is four times of the wild strain's, and its antibacterial efficiency increased obviously. The results showed that: gyrB466 amino acids had interaction with ciprofloxacin, and the mutants can reduce the bactericidal properties of ciprofloxacin.