Rapid specific detection of oral bacteria using Cas13-based SHERLOCK

ABSTRACT

Decades of ongoing research has established that oral microbial communities play a role in oral diseases such as periodontitis and caries. Yet the detection of oral bacteria and the profiling of oral polymicrobial communities currently rely on methods that are costly, slow, and technically complex, such as qPCR or next-generation sequencing. For the widescale screening of oral microorganisms suitable for point-of-care settings, there exists the need for a low-cost, rapid detection technique. Here, we tailored the novel CRISPR-Cas-based assay SHERLOCK for the species-specific detection of oral bacteria. We developed a computational pipeline capable of generating constructs suitable for SHERLOCK and experimentally validated the detection of seven oral bacteria. We achieved detection within the single-molecule range that remained specific in the presence of off-target DNA found within saliva. Further, we adapted the assay for detecting target sequences directly from unprocessed saliva samples. The results of our detection, when tested on 30 healthy human saliva samples, fully aligned with 16S rRNA sequencing. Looking forward, this method of detecting oral bacteria is highly scalable and can be easily optimized for implementation at point-of-care settings.

Keywords:

• diagnostics
• Sherlock
• rapid detection
• oral bacteria
• CRISPR-cas

Acknowledgments

We thank Dr. Xuesong He, Dr. Jeffery S. McLean, Dr. Deepak Chouhan, and Nell Spencer for their thoughtful insight and suggestions. We additionally thank Dr. Floyd Dewhirst, Dr. Mary Ellen Davey, Dr. Hey-Min Kim, Susan Yost, Stephane Viala, Christina Rothenberger, and Lujia Chen for providing bacterial strains and assistance with bacterial culturing. We were supported by the Forsyth Institute under grant number BINGHM31. We would also like to thank the Bingham Trust for their generous support of our research. This study was initiated by a Forsyth Institute and Sherlock Biosciences stragetic parnership.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

All nucleic acid sequences are provided in the manuscript or in Appx. Table A2. Raw data for Figures 1d-A4 and all code used in this project are available on Zenodo (DOI: 10.5281/zenodo.7708546). The raw data and code are also available at https://www.borlab.org/resources. Bacterial strains used in this paper will be provided upon request.

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/20002297.2023.2207336.

Additional information

Funding

The work was supported by the Forsyth Institute [BINGHM31].