Resisting cane toads through gene editing.

Since the cane toad was introduced to Queensland in 1935, it has continued to spread across northern Australia causing ecological disruption in its wake. Like other members of the family Bufonidae, cane toads produce a toxic cocktail of chemicals – “bufotoxin” – that can be lethal to many native animals that attempt to prey on them, including the northern quoll. The population of northern quolls has shown an alarming decline that has tracked the spread of toads across northern Australia since their introduction.

Within the cane toad’s natural range in Central and South America, natural predators of toads are resistant to bufotoxin due to millions of years of co-evolution. A significant proportion of this resistance is attributable to a tiny modification in a single protein – the alpha subunit of a transmembrane sodium/potassium transporter – which is encoded by either of two related genes: ATP1A1 or ATP1A3. While bufotoxin resistance could eventually arise in the northern quoll population by natural mutation after thousands of years, this will not be soon enough to protect it from its immediate threat. We hypothesised that introducing bufotoxin resistance via CRISPR-editing to the northern quoll’s ATP1A1 gene would give them an “evolutionary head-start”, then allowing natural selection to secure the species’ long-term survival.

Our project aimed to demonstrate this hypothesis first by engineering bufotoxin resistance in a related dasyurid marsupial – the fat-tailed dunnart – which we have recently established in our laboratory as a marsupial model with many of the advantages of the laboratory mouse. Our first step was to derive dunnart induced pluripotent stem cells (iPSCs), which have properties of early embryonic cells. Our recent successful derivation of dunnart iPSCs now provides us with an invaluable resource that we can use for gene editing (using CRISPR technology). Moreover, we can potentially use these cells to produce an animal carrying the gene edit, using one of several strategies that our laboratory is currently developing for marsupials. Our quoll project will continue with further funding awarded to Prof. Andrew Pask for developing the technologies necessary to de-extinct the thylacine. Having CRISPR-edited our dunnart iPSCs to introduce the bufotoxin resistance modification, our next objective is to produce a bufotoxin-resistant dunnart as a proof of principle.

In addition to deriving dunnart iPSCs, we also aimed to derive northern quoll iPSCs that could subsequently be CRISPR-edited, then used to produce a bufotoxin-resistant northern quoll. Derivation of high quality iPSCs optimally depends on first culturing fibroblast cells obtained from a very young animal (ideally embryo). Since we were only able to obtain fibroblast cells from an ageing male, we only managed to derive one iPSC line that is unlikely to be of sufficient quality. However, with our ongoing funding, we aim to continue our long-term objective of deriving new quoll iPSCs, CRISPR-editing them, and ultimately producing a bufotoxin-resistant quoll.