Rapid field-based detection of a threatened and elusive species with environmental DNA and CRISPR-Dx

With accelerating biodiversity erosion, it is critical for species conservation to use rapid and scalable monitoring methods. One powerful biodiversity monitoring method that has emerged recently is environmental DNA (eDNA). eDNA analyses currently require lengthy protocols in well-equipped laboratories, slowing down the analyses and limiting applications across broad scales. Here, we developed a protocol for eDNA analyses, leveraging CRISPR-based diagnostic systems (Dx) with lateral flow tests to rapidly process and analyze eDNA samples on site. To test the versatility of the field-based protocol, we designed a CRISPR-Dx assay specific to the threatened and elusive African manatee (Trichechus senegalensis). We sampled water across ten locations in a national park in the Republic of Congo and detected manatee DNA directly on site in almost half of the sites. We later confirmed these detections with a high-sensitivity protocol in a well-equipped laboratory. The CRISPR-Dx detections were mainly confirmed with a previously reported quantitative PCR (qPCR) assay. Despite the lower sensitivity of the field-based protocol, our analysis shows that its speed and ease of application provide advantages over slower and more expensive methods to detect threatened and elusive species, in particular if the protocol is improved in the future. Our methodology will increase the accessibility to and speed of eDNA analyses, enhancing biodiversity monitoring efforts and species conservation initiatives.