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. In vitro, the CRISPR-Cas13a system exhibited specific killing, growth inhibition, and clearance of S. Typhimurium in mixed microbial flora. In a mouse infection model, the CRISPR-Cas13a system, when delivered by a donor Escherichia coli strain, significantly reduced S. 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. IMPORTANCE 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 S. 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 in vitro and in vivo 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.

. CKPs do not mediate elimination to E. coli. We repeated the experiment shown in Fig. 2c using E. coli as the recipient. Bars represent the standard deviation of data from 3 biological replicates, dots represent each duplicate data, and P values were determined by ratio paired Student's t test     Day represents the time of the conjugation (Considered the Inoculate with d-cCKP-nontarget as day 0) and dots represent each duplicate data point.

Conjugative frequency assay in flora and proportion of S. Typhimurium during the conjugation.
E. coli Trelief5α with pUC19 (with ampicillin resistance) and S. Typhimurium (with ampicillin and tetracyclin resistance) were cultured in LB broth supplemented with 100 µg/mL ampicillin overnight. D-CKP-nontarget was cultured in LB broth supplemented with 50 µg/mL kanamycin, 34 µg/mL chloramphenicol and 0.2% glucose overnight. Strains were diluted at a ratio of 1:100 in LB broth and cultured to A600 ~ 0.5. E. coli Trelief5α and S. Typhimurium were mixed at a ratio of 1:1 to 500 µL totally. The mixture was mixed with 500 µL D-CKP-nontarget and centrifuged at 5000 × g for 5 min, LB broth was removed, and the cells were resuspended in 100 μL of 1x PBS. The 0.5 μm filters were placed on LB agar plates, and the bacterial solution was dropped on the filters for 18 hours or 36 hours. Using 1 mL PBS to wash out cells on filters. The collections were serially diluted 10-fold and cultured in LB plates (containing 0.2% glucose) supplemented with different combination of antibiotics. The number of CFUs was enumerated. Datas were calculated using the following formulas. (Amp means ampicillin, Chl means chloramphenicol, Kan means kanamycin, Tet means tetracyclin) .

Conjugtion assay in mice gut
All animal protocols were approved by the Animal Care and Use Committee of the Model Animal Research Center, College of Life Sciences, Sichuan University. Six-to eight-weekold ICR/KM mice were purchased from a local company (Dossy Experimental Animals Co., Ltd., Chengdu, China). Four mice were housed in one cage with ad libitum access to water and commercial feed. After three days of acclimation, mice were given 2 mg/ml ampicillin in their drinking water for 3 days to render them susceptible to the colonization of ampicillinresistant S. Typhimurium and then challenged with ~ 10 9 CFU of S. Typhimurium. One day later, the mice were orally inoculated with 1x10 9 CFU D-cCKP-nontarget, considered the this day as day 0, which is different from the time calculation way in Fig. 6.
Mouse feces were collected on days 1, 2, 3, 4, 5 after inoculation of D-cCKP-nontarget with a 1.5 mL centrifuge tube. The feces were weighed, and 500 mL PBS solution and 1-3 glass beads (Sangon Biotech, Shanghai, China) were added to crush the feces under a vortex mixer (Sangon Biotech, Shanghai, China). The fecal solution was serially diluted from 10 -3 to 10 -7 , plated on LB agar plates supplemented with ampicillin or with ampicillin, kanamycin, chloramphenicol and incubated at 37 °C for 16 h. The number of CFUs was enumerated. The conjugative frequency were calculated using the following formula.
= CFUs in plates with ampicillin, kanamycin, chloramphenicol CFUs in plates with ampicillin