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

Abstract

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 (shits1) mutation in B. tryoni. Genotypic analyses over three generations revealed that a high fitness cost was associated with the shits1 mutant allele and shits1 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.

Lessons from Drosophila: Engineering Genetic Sexing Strains with Temperature-Sensitive Lethality for Sterile Insect Technique Applications

“…The orthologous shibire gene has been identified in the Queensland fruit fly, Bactrocera tryoni [ 42 ], and shares approximately 90% amino acid conservation with D. melanogaster . CRISPR/Cas9 gene editing was used to introduce an identical shi ts1 mutation into B. tryoni by substituting a guanine nucleotide with adenine, which altered amino acid 268 from a highly conserved glycine residue to an aspartic acid.…”

“…A fitness cost associated with a single copy of the shi ts1 mutant allele was also evident in heterozygotes, even at a low temperature. The observed phenotype associated with the B. tryoni shi ts1 mutation was more severe than that reported for D. melanogaster, making it unsuitable to be used as a GSS tsl candidate for B. tryoni [ 42 ].…”

“…Whilst there are many advantages to the Medfly GSS, strain development has taken decades [7] and similar approaches have not produced strains for temperature sensitive sex separation in other pest species. Although the genetic basis of the Medfly tsl mutation is yet to be ascertained [39], a major facilitator superfamily gene mutation was recently found to cause the wp phenotype [40] and endeavours to identify alternative temperature sensitive mutations with the potential to generate GSSs for other pest species have been considered [41][42][43][44]. [26]).…”

Gene drive that results in addiction to a temperature-sensitive version of an essential gene triggers population collapse in Drosophila

“…In many cases, the mutations that result in TS sensitivity remain unknown, limiting the ability to move the trait into related species (59). In cases in which the nature of the TS mutation is known (as for a number of TS mutations in D. melanogaster), the ability to create similar (hopefully also TS lethal) mutations in pest species using Cas9 provides one way to simplify this process (59), though the fitness costs associated with these changes can only be discovered empirically (60).…”

Role of Genes in Regulating Host Plants Expansion in Tephritid Fruit Flies (Diptera) and Potential for RNAi-Based Control

“…For tephritid species, including Anastrepha suspense (Loew) (Diptera: Tephritidae) (Sim et al 2019), Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) (Dias et al 2019), B. dorsalis (Xie et al 2017), B. minax (Wang et al 2019), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (Cruz et al 2018), andC. capitata (Gabrieli et al 2016), effective RNAi controls have been developed based on the suppression of functional genes associated with eye pigmentation, embryonic segmentation regulation, postembryonic growth/development, reproduction, embryonic temperaturesensitive lethality and sex determination (Tayler et al 2019, Choo et al 2020). Based on these target genes, RNAi can be applied in pest control not only for tephritid species but also for some Coleopterans and Lepidoptera insects by foliar spays, ingested dsRNA or sterile insect technique (SIT) application (Baum andRoberts 2014, Spit et al 2017).…”