Bacterial Resistance to CRISPR-Cas Antimicrobials

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas based antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. Here, we systematically investigated mutations that arise from CRISPR-Cas9 induced death. We designed a genetic circuit using Cas9 from Streptococcus pyogenes (SpCas9) to study the different parameters that affect the efficiency of the system and elucidated the spontaneous genetic alterations that confer resistance to this novel type of antimicrobial agent. Our findings highlight that killing efficiency is not correlated with the type of target or number of cutting sites, however we observed that multiple target sites are valuable in order to prevent genomic mutations that can result in resistance. Furthermore, we observed that in most cases SpCas9 is the preferred target for mutations inactivating the activity of the antimicrobial by large-scale bacterial genome rearrangements involving mobile genetic elements such as insertion sequence (IS) elements. Finally, we demonstrate this can be overcome by simulating a multi-dosing strategy that resensitize escaper cells. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.