SYNCAS based CRISPR-Cas9 gene editing in predatory mites, whiteflies and stinkbugs

Abstract

Despite the establishment of CRISPR-Cas9 gene editing protocols in a wide range of organisms, genetic engineering is still challenging for many organisms due to constraints including lethality of embryo injection, difficulties in egg/embryo collection or viviparous lifestyles. Recently, an efficient CRISPR-Cas9 method, termed SYNCAS, was developed to genetically modify spider mites and thrips species. The method is based on maternal injection of formulated CRISPR-Cas9 using saponin and BAPC. Here, we investigate whether the method can be used to perform gene editing in other arthropods such as the beneficial predatory mites Amblyseius swirskii and Phytoseiulus persimilis, and the pests Bemisia tabaci and Nezara viridula. For the predatory mites, Antp and SLC25A38 were used as target genes, while the ortholog of the Drosophila melanogaster ABCG transporter white was targeted in B. tabaci and N. viridula. All species were successfully edited with the highest efficiencies (up to 39%) being obtained for B. tabaci. For A. swirskii and P. persimilis no clear phenotypes could be observed, even though SLC25A38 was successfully knocked-out. The lack of a color phenotype in SLC25A38 mutants was confirmed in the spider mite Tetranychus urticae. Disruption of the target gene Antp is likely lethal in predatory mites, as no true null mutants could be recovered. For B. tabaci, KO of white resulted in orange eyes which diverges from the phenotype seen in white mutants of D. melanogaster. In the last species, N. viridula, a single phenotypic mutant could be detected having a patchy white body coloration with wild type eye coloration. Genotyping revealed a single amino acid deletion at the target site, suggesting the creation of a hypomorphic allele. To conclude, the protocols provided in this work can contribute to the genetic study of predatory mites used in biological control, as well as hemipteran pests.