Precise single base substitution in the shibire gene by CRISPR/Cas9-mediated homology directed repair in Bactrocera tryoni

Background: Pest eradication using the Sterile Insect Technique (SIT) involves high-density releases of sterilized males that mate with wild females and ultimately suppress the population. Sterilized females are not required for SIT and their removal or separation from males prior to release remains challenging. In order to develop genetic sexing strains (GSS), conditional traits such as temperature sensitive lethality are required. Results: Here we introduce a known Drosophila melanogaster temperature sensitive embryonic lethal mutation into Bactrocera tryoni, a serious horticultural pest in Australia. A non-synonymous point mutation in the D. melanogaster gene shibire causes embryonic lethality at 29 °C and we successfully used CRISPR/Cas9 technology to recreate the orthologous shibire temperature sensitive-1 (shi(ts1)) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shi(ts)1 mutant allele and shi(ts1) homozygotes were not viable at 21 °C, which is a more severe phenotype than that documented in D. melanogaster. Conclusions: We have demonstrated the first successful use of CRISPR/Cas9 to introduce precise single base substitutions in an endogenous gene via homology-directed repair in an agricultural pest insect and this technology can be used to trial other conditional mutations for the ultimate aim of generating genetic sexing strains for SIT.Amanda Choo, Elisabeth Fung, Isabel Y. Chen, Robert Saint, Peter Crisp and Simon W. Baxte

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