Bacterial resistance to CRISPR-Cas antimicrobials

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas 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. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations.

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Fig: gRNA targets in the chromosome of E. coli K-12 MG1655. (a) Chromosomal map of E. coli K-12 MG1655 indicating the oriC and the cutting sites of the different CRISPR targets (CRTs). (b) Efficiency of the CRTs determined by the surviving fraction, the CRTs are ordered by gRNAs targeting non-coding vs. coding regions, non-essential vs. essential genes and the number of cutting sites. CRT0 indicated as a black bar served as a control gRNA with a non-targeting sequence.

We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.

Uribe, R.V., Rathmer, C., Jahn, L.J. et al. Bacterial resistance to CRISPR-Cas antimicrobials. Sci Rep 11, 17267 (2021). https://doi.org/10.1038/s41598-021-96735-4

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