Abstract
We demonstrate that simple, non-invasive environmental DNA (eDNA) methods can detect transgenes of genetically modified (GM) animals from terrestrial and aquatic sources in invertebrate and vertebrate systems. We detected transgenic fragments between 82–234 bp through targeted PCR amplification of environmental DNA extracted from food media of GM fruit flies (Drosophila melanogaster), feces, urine, and saliva of GM laboratory mice (Mus musculus), and aquarium water of GM tetra fish (Gymnocorymbus ternetzi). With rapidly growing accessibility of genome-editing technologies such as CRISPR, the prevalence and diversity of GM animals will increase dramatically. GM animals have already been released into the wild with more releases planned in the future. eDNA methods have the potential to address the critical need for sensitive, accurate, and cost-effective detection and monitoring of GM animals and their transgenes in nature.
Highlights
Environmental DNA is DNA extracted from environmental samples such as soil, sediment, water, air, feces, dust, as well as bulk DNA from artificial and natural collectors like Malaise insect traps, ocean sponges, and spider webs [1–4]. eDNA techniques commonly employ polymerase chain reactions (PCR), qPCR, and recently ddPCR to amplify taxonomically informatic DNA markers including 16S and 18S rRNA, cytochrome c oxidase I (COI), and the internal transcribed spacer (ITS) from traces of DNA found in the environment for detection of specific species [5, 6]
We report that fragments of transgenes from genetically modified (GM) animals are detectable noninvasively via environmental DNA across three different animal systems: invertebrates, mammals, and fish (Fig 1)
Transgene identities of aligned amplicons were confirmed by NCBI BLAST using default settings and alignment with reference genes downloaded from the GenBank Nucleotide database [31]
Summary
Environmental DNA (eDNA) is DNA extracted from environmental samples such as soil, sediment, water, air, feces, dust, as well as bulk DNA from artificial and natural collectors like Malaise insect traps, ocean sponges, and spider webs [1–4]. eDNA techniques commonly employ PCR, qPCR, and recently ddPCR to amplify taxonomically informatic DNA markers including 16S and 18S rRNA, cytochrome c oxidase I (COI), and the internal transcribed spacer (ITS) from traces of DNA found in the environment for detection of specific species [5, 6]. High-throughput next-generation sequencing of DNA markers and shotgun sequencing have been utilized to generate large genetic data sets that span across taxonomic groups for community-level studies [9–13] These eDNA methods have revolutionized biodiversity research and are increasingly used by academic biologists, environmental regulatory agencies, and private industry for biomonitoring purposes [14]. In parallel to the development of eDNA methods for biomonitoring, the advent of CRISPRbased genome-editing technologies have revolutionized molecular biology by vastly simplifying the process of creating genetically modified (GM) organisms, which has allowed transgenic research and production to advance dramatically [15]. This sudden democratization of genome-editing is leading to an explosion in the diversity of genetic modifications, the kinds of species targeted, and the contexts in which these methods are applied [16]. We report that fragments of transgenes from GM animals are detectable noninvasively via environmental DNA across three different animal systems: invertebrates (fruit flies; Drosophila melanogaster), mammals (laboratory mice; Mus musculus), and fish (black tetras; Gymnocorymbus ternetzi) (Fig 1)
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