Bacterial resistance to CRISPR-Cas antimicrobials

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Antibiotic resistance necessitates novel therapeutic strategies.
• CRISPR-Cas antimicrobials offer precise targeting but require understanding resistance mechanisms.
• Engineering the microbiome relies on effective and robust antimicrobial agents.

Purpose of the Study:

• To investigate how E. coli develops resistance to episomally-encoded CRISPR-Cas9 antimicrobials.
• To elucidate the genetic alterations underlying CRISPR-Cas9 resistance.
• To identify strategies for enhancing CRISPR-Cas antimicrobial efficacy and reducing resistance.

Main Methods:

• Systematic investigation of CRISPR-Cas9 killing efficiency against a target E. coli strain.
• Analysis of resistance mutation rates and identification of genetic alterations.
• Utilizing Streptococcus pyogenes Cas9 (SpCas9) for antimicrobial activity assessment.

Main Results:

• CRISPR-Cas9 killing efficiency is independent of the number of cutting sites or target type.
• Increasing the number of targets reduced chromosomal mutations conferring resistance.
• The primary resistance mechanism involved bacterial genome rearrangements inactivating plasmid-encoded SpCas9 via mobile genetic elements.

Conclusions:

• CRISPR-Cas9 resistance in E. coli is frequently mediated by SpCas9 inactivation through genome rearrangements.
• Resistance mechanisms can be overcome by re-introducing functional SpCas9.
• Findings guide the design of more effective CRISPR-Cas antimicrobials with reduced resistance emergence.