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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  ts1  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.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12863-020-00934-3.

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