Gut-adapted bacteria such as probiotic strains could prove more resilient than bacteriophages within the intestinal microbiota and use bacterial conjugation to deliver CRISPR- cas9 to a broader range of target cells. Furthermore, phage receptors on the cell surface can quickly mutate (Shabbir et al, 2016) and environmental conditions (low pH, gastric fluid, proteases, etc.) encountered by viral particles can dramatically limit their activity in the gut (Nobrega et al, 2016). While bacteriophages offer interesting advantages such as high infectivity, they often display narrow host ranges (Yosef et al, 2015). Engineered bacteriophages have been proposed as delivery vehicles for CRISPR- cas9 systems, and proof-of-concept demonstrations have been performed in bacterial cultures as well as in mice models (Bikard et al, 2014b Citorik et al, 2014 Yosef et al, 2015 Hsu et al, 2020). The key step to enable this technology for antimicrobial applications is to deliver the CRISPR- cas9 system to virtually all bacteria of the targeted population. CRISPR-Cas9 cleavage of a chromosomal sequence leads to the death of the target bacterium (Gomaa et al, 2014 Cui & Bikard, 2016), whereas targeting a sequence found on a plasmid leads to its loss from the host cell (Dong et al, 2019 Neil et al, 2019). Upon detection of a valid protospacer in the genome of the target bacterium, the CRISPR-Cas9 complex induces a double-strand cut that fails to be repaired by non-homologous end joining mechanisms as shown in E. coli (Mali et al, 2013 Cui & Bikard, 2016).
The specificity of this system relies on the complementarity between a 20-nucleotide sequence (spacer) present in a guide RNA (gRNA) and a target DNA sequence (protospacer) sitting next to a protospacer adjacent motif (PAM) (Koonin & Makarova, 2019). CRISPR-Cas9 is a protein–RNA complex that can be programmed to cleave specific DNA sequences found only in target bacteria. The use of CRISPR-Cas9 as an antimicrobial represents a promising strategy to precisely eliminate targeted bacterial populations. Among these pathogens, E. coli is responsible for various diseases in farm animals and accounts for almost half of all human antibiotic-resistant infections attributed to enterobacteria (Cassini et al, 2019). This group comprises pathogens such as Escherichia coli, Klebsiella pneumoniae, Shigella sp., and Salmonella sp., which can cause deadly infections when not adequately treated (Falagas et al, 2014). Enterobacteriaceae are natural residents of the gut microbiota and are particularly infamous for their rapid accumulation of antibiotic resistance genes (Nordmann et al, 2011 Yamamoto & Pop-vicas, 2014). Rates of antibiotic-resistant infections are rapidly increasing worldwide and threaten to become the second most important cause of mortality by 2050 (World Health Organization, 2014).