CRISPR/Cas12a DNA detection of Borrelia burgdorferi in black-legged ticks (Ixodes scapularis)

CRISPR/Cas systems may revolutionize DNA detection by providing a sensitive and user-friendly approach. With CRISPR/Cas DNA detection a ribonucleoprotein, comprised of a guide RNA and Cas12a enzyme, binds to and cuts DNA targets with high specificity. Following cutting, the Cas12a enzyme exhibits nonspecific collateral cleavage of single-stranded DNA, which can be harnessed to cleave a fluorescent reporter molecule. We developed a CRISPR/Cas12a detection protocol to identify Borrelia burgdorferi, the Lyme disease pathogen, in black-legged ticks (Ixodes scapularis) collected from forests in Rutland County, Vermont. We compared CRISPR/Cas12a detection of PCR products on a fluorescent plate reader to gel electrophoresis detection. We further compared this detection system to quantitative real-time PCR (qPCR) using a different B. burgdorferi-specific primer set.
Results of B. burgdorferi infection were nearly identical among all three tests. qPCR exhibited the highest sensitivity, likely because we used a higher cycle number with this test (40 qPCR cycles vs. 30 endpoint PCR cycles). We found 118/126 (94%) agreement between the results of gel electrophoresis and CRISPR/Cas detection of PCR amplification products. We found six samples that were positive with the CRISPR/Cas12a test but negative with gel electrophoresis. Five of these samples were negative by qPCR, suggesting improvements are needed to the interpretation of the plate reader output. CRISPR/Cas detection of B. burgdorferi is more convenient than gel electrophoresis and the highly specific test has the potential to be applied to strain-level analyses.