Escape from antimicrobial CRISPR-Cas9 in E. coli ST131 depends on the genetic context of the target gene.

Escherichia coli (E. coli) is a common bacterium in the human gut and an important cause of intestinal and extraintestinal infections. Some E. coli sequence types (ST) are associated with high pathogenicity. The Extraintestinal Pathogenic E. coli (ExPEC) ST131 is a globally distributed multidrug-resistant human pathogen associated with urinary tract and bloodstream infections. Antibiotic-resistant infections often lead to antibiotic treatment failure, underscoring the need of developing alternative treatments. The highly selective antimicrobial potential of CRISPR-Cas9 has been demonstrated in a range of model organisms. However, the effectiveness of CRISPR-Cas9 in combating ST131-associated infections and the consequences of CRISPR-Cas9 treatment, such as the emergence of escapers, remains unclear. Here, we investigated the antimicrobial activity of CRISPR-Cas9 against ST131 and assessed the frequency and genetic basis of escape. We conjugatively delivered CRISPR-Cas9 to ST131 isolates which carried cefotaxime-resistance-encoding target gene blaCTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: blaCTX-M-15-positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a [~]10-5 frequency. Instead, blaCTX-M-15-negative escapers presented chromosomal deletions involving blaCTX-M-15 loss. The frequency of blaCTX-M-15 loss depended on the blaCTX-M-15 genetic context. Specifically, blaCTX-M-15-negative escapers emerged at low frequency ([~]10-5) in isolates where blaCTX-M-15 was located downstream of insertion sequence (IS) ISEcp1, while escapers emerged with high frequency ([~]10-3) in isolates where blaCTX-M-15 was flanked by IS26. This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS26 may drive increased frequencies of escape. IMPORTANCEIn the past decade CRISPR-Cas9 has emerged as an efficient antimicrobial tool capable of selective elimination of targeted bacteria. Even though it has been well described that bacteria can evolve to escape targeting by CRISPR-Cas9, the mechanisms of bacterial escape and their consequences remain largely elusive. In this study, we demonstrate the antimicrobial efficacy of CRISPR-Cas9 against natural isolates of Escherichia coli ST131, a clinically relevant pathogen, and elucidate the mechanism of escape from antimicrobial activity. We identify two distinct mechanisms of escape, which involve either dysfunctional CRISPR-Cas9 activity, or loss of the target gene (blaCTX-M-15), with the latter occurring at frequencies that depend on the genetic context of the target gene. These findings provide important insights into the frequency and mechanisms of bacterial escape from CRISPR-Cas9-based antimicrobials and offer a foundation for the development of more effective treatments.