Genetically engineered resistance to bufotoxin in marsupial ATP1A1

The introduction of the bufotoxin-secreting cane toad ( Rhinella marina ) to Queensland in 1935 has had a devastating impact on wildlife in the Australian tropics. Having evolved for millions of years in the absence of cane toads or other bufotoxin-secreting organisms, many of the Australia’s native predators that include cane toads in their diet suffered large population declines following cane toad invasion to their habitat. One marsupial species, the northern quoll ( Dasyurus hallucatus ), is now classified as endangered (IUCN Red List) largely due to bufotoxin ingestion. This study aimed to introduce bufotoxin resistance into a marsupial cell line by editing part of the ATP1A1 gene encoding the extracellular H1-H2 domain – the binding target of bufotoxin. To this end, CRISPR prime editing was used to replace the part of the wildtype ATP1A1 gene encoding the H1-H2 domain in fibroblasts of a related marsupial model, the fat-tailed dunnart ( Sminthopsis crassicaudata ), with modifications known to be associated with bufotoxin resistance. The genetically modified cell population showed a >45-fold increase in resistance to bufalin (an active component of bufotoxin) compared to wild type. This study provides a proof of concept towards engineering genetic resistance in the northern quoll to halt or even reverse its current population decline.