The Role of DNA Repair Systems in Antibiotic Resistance Acquisition: Using CRISPR-Cas9 to Compare HDR and MMEJ in Escherichia coli K-12

Abstract

Antibiotic resistance is a major challenge in healthcare. A better understanding of how bacteria can develop antibiotic resistance is needed in order to develop more effective antibiotics. This study investigates how bacterial DNA repair mechanisms might contribute to antibiotic resistance, using E. coli K12 and the antibiotic streptomycin as a model system. When E. coli experience double-stranded breaks in their DNA, they repair the damage using either homology-directed repair (HDR), which uses a DNA template to repair the break, or microhomology-mediated end joining (MMEJ), which often causes random mutations by using small, non-homologous DNA segments. These two repair mechanisms were examined in the rpsL gene, which encodes a protein in E. coli's 30S ribosomal subunit, the target of streptomycin and other aminoglycoside antibiotics. Using CRISPR-Cas9 technology, targeted double-stranded breaks were induced in the rpsL gene of E. coli, and two distinct repair approaches were tested: (a) template-present editing: using HDR to insert a synthetic template containing a K43T resistance-conferring mutation as a positive control, and (b) template-absent editing: forcing MMEJ to create mutations. The bacteria were then grown on streptomycin media to assess resistance development. Template-present editing produced many resistant colonies, confirming the effects of the K43T mutation. More importantly, template-absent modification produced a limited number of resistant colonies, demonstrating that natural MMEJ-induced mutations in the rpsL gene can confer antibiotic resistance. These findings improve our understanding of how DNA repair mechanisms can inadvertently lead to antibiotic resistance and provide crucial insights for developing more effective treatments and monitoring strategies.