Inhibition of horizontal kanamycin-resistance gene transfer using specific CRISPR/Cas9 system

Abstract

Objective To explore an effective technique to control the horizontal transfer of drug-resistance genes using specific CRISPR/Cas9 system. Methods Three pairs of kanamycin-resistance gene (Kan)-specific single guide RNAs (sgRNAs) were designed, annealed and inserted into the Bsa Ⅰ site of pCas9, respectively. Each group of recombinant bacteria competent cells were transformed by pET-30a that carried Kan gene. The transformants were spread on plates containing chloramphenicol or chloramphenicol/kanamycin to calculate and compare the transformation efficiencies. The DH5α competent cells containing pET-30a were transformed with each recombinant plasmid to detect the degradation of Kan gene mediated by specific sgRNAs with PCR and to observe the effects on growth curve of recombinant. Results The transformation efficiencies of Kan gene into Escherichia coli strains harboring specific sgRNAs CRISPR/Cas9 system decreased 100%-88.08%. The specific sgRNAs CRISPR/Cas9 system degraded Kan gene without affecting the growth of bacteria. Conclusion The specific CRISPR/Cas9 system inhibits the horizontal transfer of bacterial kanamycin-resistance gene. Key words: CRISPR/Cas9 system; Drug resistance; Horizontal gene transfer