The Comparative Nature of CRISPRi and Antibiotic Interaction

Abstract

Clinically, the use of multi-drug therapies that lead to synergistic effects can lower the necessary drug dosage and can reduce the ability for bacteria or viruses to evolve resistance to drugs. Yet, even though drug synergy is becoming more relevant in treating patients, there is still a lack of a simple method to detect novel synergistic pairs. Here we propose a method, is a method proposed to identify synergetic drug pairs in Escherichia coli using a duplexed CRISPR interference (CRISPRi). We used duplexed CRISPRi to simultaneously reduce transcription of a gene target pair (folA and folP) by a known synergistic antibiotic pair (sulfamethoxazole and trimethoprim), and found that the simultaneous transcriptional repression also led to synergistic effects. To demonstrate whether our duplexed system can predict synergistic or additive effects caused by transcription expression in antibiotic-targeted genes, we used three antibiotic gene targets who have known antibiotic interaction types when used in combination. The antibiotic chosen targets the biosynthesis of folate; MAC 173979(MAC, gene target pabB), Sulfamethoxazole(SMX, gene target folA), Trimethoprim(TMP gene target folP). SMX-TMP are known in literature to be synergistic in E.coli and SMX-MAC and TMP-MAC are known to be additive. We designed 3 plasmids with duplex sgRNA insertion to match each combination using the gene targets of the antibiotics. As a result, the folA-folP showed strong synergistic effects (p=0.0092), In comparison, we also demonstrated that there is no synergistic effect when simultaneously reducing the expression of folA or folP with pabB, a gene target of the antibiotic MAC173979, consistent with previous data from antibiotic experiments.Together, our data here on pairwise combinations from 3 antibiotic gene targets show that duplexed CRISPRi is a promising method to detect synergistic antibiotic target pairs.