Evaluation and Application of CRISPR-Cas12a-based Systems for Amphibian Detection from Dietary and Environmental DNA

Current methods for monitoring biodiversity are hindered by detection efficiencies or rely on expensive laboratory equipment. The goal of this study was to evaluate the application of a CRISPR-Cas12a-based system for the species-specific detection of amphibians from dietary and environmental DNA samples. A Cas-12a-based platform was designed and analyzed for the fluorescent detection of three salamander species; Desmognathus fuscus, D. ochropheaus, and Erycia bislineata. This system was applied to the identification of salamander DNA extracted from the fecal sacs of nestling Louisiana Waterthrush (Parkesia monticola), amplified with PCR primers targeting the CYT B mtDNA gene, and compared to DNA metabarcoding. A similar approach to detect five species of forest-dependent Borneo frogs was evaluated in-silico. These studies indicate that this fluorescent-based detection approach using CRISPR-CAS12a can be a reliable method for multiple species detection at remote field stations that are lacking in sequencing technology