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Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in <i>Enterobacteriaceae</i> is mainly due to the metalloenzyme FosA3 encoded by the <i>fosA3</i> gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a <i>fosA3⁺</i>  <i>Escherichia coli</i> strain. The <i>fosA3⁺</i>  <i>E. coli</i> strain was generated by transforming synthetic <i>fosA3</i> into a nonpathogenic <i>E. coli</i> TOP10. To mediate the <i>fosA3</i> disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the <i>fosA3</i> sequence of more than 700 <i>fosA3</i>⁺  <i>E. coli</i> isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic <i>fosA3</i> gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against <i>fosA3⁺</i> bacteria.