Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii
Abstract Background: The invasive fruit pest Drosophila suzukii has been reported for the first time in Europe and 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 has opened the doors for researchers to develop novel biotechnologically based pest control approaches. Stage and or tissue specific 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 diseases vectors for sexing, monitoring and reproductive biology studies. Here, we demonstrate an improvement in the efficiency of CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system.Results: To 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. As a comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. To evaluate the homology-dependent repair (HDR)-based genome editing efficiency, we used as a target platform a previously established transgenic line that expresses DsRed ubiquitously. In addition, we isolated 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.Conclusion: The use of the endogenous promoters of D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, showed improved efficiency of gene editing compared to previous reports. 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. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.