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 bla CTX-M-15 in the chromosome and characterized escape subpopulations. Two main types of escapers emerged: bla CTX-M-15 -positive escapers carried dysfunctional CRISPR-Cas9 systems and arose at a ~10 -5 frequency. Instead, bla CTX-M-15 -negative escapers presented chromosomal deletions involving bla CTX-M-15 loss. The frequency of bla CTX-M-15 loss depended on the bla CTX-M-15 genetic context. Specifically, bla CTX-M-15 -negative escapers emerged at low frequency (~10 -5 ) in isolates where bla CTX-M-15 was located downstream of insertion sequence (IS) IS Ecp1 , while escapers emerged with high frequency (~10 -3 ) in isolates where bla CTX-M-15 was flanked by IS 26 . This work emphasizes how the genetic context of target genes can drive the outcome of CRISPR-Cas9 tools, where the presence of IS 26 may drive increased frequencies of escape.