Multiplexed deactivated CRISPR-Cas9 gene expression perturbations deter bacterial adaptation by inducing negative...

Area of Science:

• Microbiology
• Genetics
• Drug Discovery

Background:

• The rise of multi-drug resistant bacteria and a dwindling antibiotic pipeline demand novel strategies to combat antibiotic resistance.
• Microorganisms' inherent adaptability poses a significant challenge to existing treatments.

Purpose of the Study:

• To develop a novel therapeutic strategy to slow the evolution of antibiotic resistance by inducing epistasis between gene perturbations.
• To investigate the efficacy of the Controlled Hindrance of Adaptation of OrganismS (CHAOS) approach in preventing bacterial adaptation.

Main Methods:

• Constructed a combinatorial library of multiplexed, deactivated CRISPR-Cas9 devices to perturb gene expression in *Escherichia coli*.
• Assessed the fitness and adaptive potential of engineered strains during antibiotic exposure.
• Utilized multiplexed peptide nucleic acids to enhance antibiotic susceptibility in clinical isolates.

Main Results:

• Multiplexed gene perturbations led to significant fitness loss via epistasis, unlike individual perturbations.
• Strains exhibiting epistasis demonstrated significantly slower adaptation over 3-14 days, with sustainable loss in adaptive potential.
• Multiplexed peptide nucleic acids increased antibiotic susceptibility in carbapenem-resistant *E. coli* through epistasis.

Conclusions:

• The CHAOS approach, by inducing epistasis, offers a promising new therapeutic strategy to restrict the evolution of antibiotic resistance.
• This method effectively deters bacterial adaptation and resensitizes resistant strains to antibiotics, addressing a critical global health challenge.