Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina)

Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia’s most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.

• Publication year

  2025

• Venue

  bioRxiv

• Publication date

  2025-05-16

• Fields of study

  Biology, Medicine, Environmental Science

• Identifiers
  DOI 10.1177/25731599251382427PMID 41002041
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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• Progress in invasion biology: predicting invaders.

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