Detection of the jellyfish Chrysaora pacifica by RPA-CRISPR-Cas12a environmental DNA (eDNA) assay and its evaluation through field validation and comparative eDNA analyses

Abstract

Climate-driven environmental changes and anthropogenic activities can result in the proliferation of non-indigenous aquatic species such as jellyfish that may cause envenomation and various ecological disruptions. Here we developed a two-step RPA-CRISPR-Cas12a eDNA assay, consisting of target eDNA amplification followed by a CRISPR-Cas12 reaction, for the early detection of Chrysaora pacifica, a jellyfish species often considered non-indigenous to South Korea. The assay demonstrated high sensitivity, with a detection limit of two copies COI/μL for eDNA derived from C. pacifica, using target specific RPA primers and crRNA sequences. Field validation of the assay using eDNA samples from Jinhae Bay collected over eight months of time-series monitoring, revealed temporal distribution of the jellyfish which correlated with results of digital polymerase chain reaction (dPCR) and eDNA metabarcoding. The C. pacifica eDNA assays were also corroborated (R-square 0.7891) by reports from a citizen science–based jellyfish-monitoring program operated by the National Institute of Fisheries Science, South Korea. Our RPA-CRISPR-Cas eDNA assay can therefore, be an efficient alternative to traditional tools for the early detection of outbreaks of non-indigenous or harmful species in marine ecosystems.