Pathogen-Specific Bactericidal Method Mediated by Conjugative Delivery of CRISPR-Cas13a Targeting Bacterial Endogenous Transcripts

Abstract

The emergence of antibiotic-resistant bacteria threatens public health, and the use of broad-spectrum antibiotics often leads to unintended consequences, including disturbing the beneficial gut microbiota and resulting in secondary diseases. Therefore, developing a novel strategy that specifically kills pathogens without affecting the residential microbiota is desirable and urgently needed. Here, we report the development of a precise bactericidal system by taking advantage of CRISPR-Cas13a targeting endogenous transcripts of Salmonella enterica serovar Typhimurium delivered through a conjugative vehicle. <i>In vitro</i>, the CRISPR-Cas13a system exhibited specific killing, growth inhibition, and clearance of <i>S.</i> Typhimurium in mixed microbial flora. In a mouse infection model, the CRISPR-Cas13a system, when delivered by a donor Escherichia coli strain, significantly reduced <i>S.</i> Typhimurium colonization in the intestinal tract. Overall, the results demonstrate the feasibility and efficacy of the designed CRISPR-Cas13a system in selective killing of pathogens and broaden the utility of conjugation-based delivery of bactericidal approaches. <b>IMPORTANCE</b> Antibiotics with broad-spectrum activities are known to disturb both pathogens and beneficial gut microbiota and cause many undesired side effects, prompting increased interest in developing therapies that specifically eliminate pathogenic bacteria without damaging gut resident flora. To achieve this goal, we developed a strategy utilizing bacterial conjugation to deliver CRISPR-Cas13a programmed to specifically kill <i>S.</i> Typhimurium. This system produced pathogen-specific killing based on CRISPR RNA (crRNAs) targeting endogenous transcripts in pathogens and was shown to be effective in both <i>in vitro</i> and <i>in vivo</i> experiments. Additionally, the system can be readily delivered by conjugation and is adaptable for targeting different pathogens. With further optimization and improvement, the system has the potential to be used for biotherapy and microbial community modification.