# CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples

## TL;DR

This paper explores using CRISPR/Cas12 technology to detect Borrelia bacteria in human samples, offering a potentially more accessible alternative to traditional PCR methods.

## Contribution

The study introduces a CRISPR/Cas12-based detection system for Borrelia sensu lato and compares its performance with real-time PCR.

## Key findings

- The CRISPR/Cas12 system reliably detected Borrelia DNA at low concentrations when combined with PCR.
- Detection sensitivity decreased in the presence of human genomic DNA.
- End-point PCR improved detection for certain Borrelia genospecies but required more cycles.

## Abstract

Background/Objectives: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR. Methods: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR. Results: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA. Conclusions: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.