Therapeutic Overview

The most effective treatment for Lyme disease is currently entry-level antibiotics, typically doxycycline (CDC, 2023). However, this is not an ideal therapeutic, as it contributes to antimicrobial resistance and fails to effectively address the rising numbers of post-treatment Lyme disease syndrome (PTLD). Recent studies have shown proof of antibiotic tolerance and resistance among Borrelia burgdorferi strains, and this will cause current treatments to become increasingly ineffective (Bobe et al., 2021). This issue, combined with delayed diagnostics, is increasing current and predicted cases of PTLD (Hirsch et al., 2020). With LANCET, Lambert iGEM allows for patients to be both diagnosed and treated efficiently, allowing for minimal possible complications in relation to resistance, and resulting in a lower possibility of chronic symptoms (see Diagnostic Overview).

Our novel therapeutic builds upon our 2024 project, SHIELD, which primarily focused on the creation of an antibiotic alternative to combat AMR. This approach utilizes the CRISPR-interference (CRISPRi) system, consisting of a deactivated Cas9 protein and sgRNA, to downregulate critical genes in resistant bacteria. As a part of LANCET, the system was redesigned to target the critical genes Bb0250 and Bb0841 in the Borrelia burgdorferi bacteria. In addition to preliminary testing as done the previous year, we successfully used the multiplexed system, tested it in vivo, and created a lipid nanoparticle testing plan.

Preliminary Testing

Utilizing the CRISPRi protocol optimized in SHIELD, we individually tested all the sgRNAs designed for each gene to experimentally determine which one exhibited the greatest repression. Each reaction was completed in cell-free systems, and a Syntek plate reader was used to record fluorescent output over time. Of the sgRNAs, 60.5 and 54.3 repressed most efficiently for Bb0250 and Bb0841, respectively, at an average fluorescent decrease of 72.59% and 65.12% (see Fig. 1).

Figure 1. Repression of validated sgRNAs were 72.59% and 65.12% for genes Bb0250 and Bb0841 respectively.

Multiplexed Reaction

A multiplexed system targets multiple genes simultaneously, potentially allowing for a greater net repression of gene activity and overall bacterial death. The most repressive sgRNAs for individual genes were combined into a single well, with the reaction protocol outlined in experimentation (see. Fig 2). The results of this represent how the bacteria’s critical functions would be further limited by the repression of multiple genes simultaneously.

Figure 2. Average of triplicate reactions show 76.01% repression overall, showing sufficient repression of combined CRISPRi systems.

In vivo Experimentation

To further prove the efficacy of our system, we moved to testing in vivo, using Escherichia coli as a proof of concept. We transformed DH5 Alpha E. coli competent cells with our plasmid, including the dCas9 protein and sgRNA targeting the rpsL gene. Using the LIVE/DEAD™ BacLight™ Bacterial Viability Kit from Thermofisher, we performed a bacterial viability assay and visualized evidence of cell death as a result of repression. The sample transformed with both dCas9 and sgRNA saw about a 9.5-fold decrease in viable biomass than when transformed with just dCas9, and a near 14-fold increase in the proportion of dead to alive cells, with over 60% of the population marked as dead (see Figs. 3-4).

Figure 3. Average Green RFU for dCas9 alone was 1,005,200, and 105,466.67 for dCas9-sgRNA, showing a 9.53-fold decrease for the experimental group, and proving the preventative effect of the CRISPRi.

Figure 4. Average dead/alive ratio for dCas9 alone was 0.219, and 3.13 for dCas9-sgRNA, a 14.26-fold increase in experimental dead/alive bacteria. The dCas9-sgRNA group also showed the dead cell signal constituting 60.7% of the total, with dCas alone presenting just 17.7%..

Lipid Nanoparticles

Lipid nanoparticles were chosen as a viable and safe delivery mechanism for CRISPRi, due to its recently proved efficacy in successfully delivering DNA, and relatively low cytotoxicity. The specific formulation and ratio of lipids in an LNP are critical in optimizing the encapsulation and delivery of different plasmids; in this case, the pdCas9sgRNA. Based on existing literature, we compiled data from extensively tested LNPs to create a protocol and LNP formulation most optimal for our own system (see LNP).

Planned LNP Workstream (summary):

• Select formulation and lipid ratios optimized for plasmid (pdCas9sgRNA) encapsulation.
• Benchmark transfection/uptake vs. controls in relevant models.
• Assess cytotoxicity and delivery efficiency across candidate LNP recipes.
• Iterate composition based on delivery + viability metrics.

References