Beach, S. J. | Search bioRxiv

bioRxiv2025

Efficient CRISPR-Cas9-mediated genome editing of the cane toad (Rhinella marina)

Invasive species inflict major ecological, economic, social, and cultural harm worldwide, highlighting the urgent need for innovative and effective control strategies. Genome editing offers exciting possibilities for creating highly targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australias most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes that provide clear 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, where we achieved maternal germline transmission rates of almost 100%. This technology, representing the first application of CRISPR-Cas9 in the Bufonidae family, opens possibilities for exploring both basic research questions and strategies for population control.

synthetic biology↗

bioRxiv2024

Recombinant venom proteins in insect seminal fluid reduces female lifespan

The emergence of insecticide resistance has increased the need for alternative pest management tools1,2 Numerous genetic biocontrol approaches, which involve the release of genetically modified organisms to control pest populations, are in various stages of development to provide highly targeted pest control3-7. However, all current mating-based genetic biocontrol technologies function by releasing engineered males which skew sex-ratios or reduce offspring viability in subsequent generations. This allows mated females continue to cause harm (e.g. transmit disease). Here, we demonstrate the first example of intragenerational genetic biocontrol, wherein mating with engineered males reduces female lifespan. The toxic male technique (TMT) involves the heterologous expression of insecticidal proteins within the male reproductive tract that are transferred to females via mating. We demonstrate TMT in Drosophila melanogaster males, which reduce the median lifespan of mated females by 37 - 59% compared to controls mated to wild type males. Agent-based models of Aedes aegypti predict that compared to existing genetic biocontrol technologies, even modest levels of mated female mortality could allow TMT to suppress a female population substantially faster, which is likely to result in reduced disease burdens. TMT presents a novel approach for combatting outbreaks of disease vectors and agricultural pests. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/575309v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@9930eforg.highwire.dtl.DTLVardef@15bbc0borg.highwire.dtl.DTLVardef@1967af9org.highwire.dtl.DTLVardef@63a589_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗