CRISPR-Dx and Metabarcoding Perform Similarly for Monitoring Mammals With eDNA on the Catchment Level in High-Alpine Ecosystems

Biodiversity monitoring in difficult-to-access areas, such as rugged mountain ranges, is currently challenging and thus often absent. Environmental DNA (eDNA) offers new opportunities to monitor remote or strictly protected areas, as rivers integrate the biodiversity information of entire catchments. Environmental samples can be analyzed either with metabarcoding or using species-specific assays. Species-specific assays like quantitative polymerase chain reaction assays do not require a fully-equipped laboratory and thus can be used in settings with limited resources and are especially suited to monitoring elusive or threatened species of management concern. Recently developed molecular tools, such as CRISPR-based diagnostic systems (CRISPR-Dx), provide new avenues to facilitate eDNA analysis through species-specific assays. Here, we combine multispecies primers with CRISPR-Dx to detect terrestrial mammal species in parallel with one amplification to detect multiple species with CRISPR-Dx. Given the short length of metabarcoding amplicons, designing species-specific assays within them can be challenging. We designed species-specific CRISPR-Dx for eight terrestrial mammals within a commonly used metabarcoding amplicon ~59 base pairs in length and tested the assays on eDNA samples collected in high-alpine catchments. Additionally, we compared the detections from CRISPR-Dx with metabarcoding results of the same samples and with catchment-based species inventories obtained through traditional monitoring. First, we show that designing species-specific CRISPR-Dx within a short amplicon allows terrestrial mammal detection in eDNA. Second, we demonstrate that CRISPR-Dx assays combined with multispecies primers are comparable in sensitivity to metabarcoding and thus can bridge a gap between species-specific assays and community analysis without requiring fully equipped laboratories. Third, we highlight that catchment-based eDNA sampling can be used to monitor terrestrial mammals in remote or protected areas. Overall, we demonstrate that eDNA and particularly CRISPR-Dx are a promising tool to monitor inaccessible and/or protected areas and to detect rare species across large spatiotemporal scales, thereby promoting biodiversity conservation.