Abstract
BackgroundThe invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.ResultsTo improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing.ConclusionThe deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.
Highlights
The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since
Improvement on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) genome editing in Drosophila suzukii In order to improve on the homology-dependent repair (HDR)-mediated genome editing based on CRISPR/Cas9-induced double strands break (DSB), we isolated endogenous polymerase II and polymerase III promoters (U6 genes) from D. suzukii to drive Cas9 or Guide RNA (gRNA), respectively
Searching for homologs of the D. melanogaster heat shock protein 70 gene, we identified the D. suzukii Ds_hsp70 gene, cloned and sequenced 500 bp upstream of the ATG translation start codon and used this upstream sequence to drive the expression of Cas9
Summary
The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since . The system has been proposed more than half a century ago and was used to successfully eradicate the tsetse fly from Zanzibar as well as the screw worm from Libya and the USA [17, 18] It encompasses mass production of the target insect, removal of the females, and sterilization of males by ionizing radiation prior to release [16]. For monitoring the competitiveness of released males, sperm-marking systems were developed for a number of pest insects and diseases vectors by driving the expression of fluorescent protein during spermatogenesis [23,24,25,26]
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