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 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 diseases 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. 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. 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. Conclusion: The 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. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

“…The vector piggyBac has gained particular attention due to its versatility and usability in different systems. When we started to use piggyBac for germline transformation of an Italian strain of D. suzukii, we had only poor success and retrieved a rare transgenic line (06_F5M2) carrying construct HMMA006 [52], which mediates early embryonic expression of tTA ( Fig. 1), with a transformation rate of 1.6% (300 embryos injected, 200 survived, 60 fertile, 1 transgenic line).…”

“…To examine, whether the 300 bp upstream regulatory element plus the 49 bp 5'UTR drive cellularization-specific gene expression, we fused this 349 bp enhancer/promoter fragment of the Ds_sry α gene to tTA (Fig. 1b) and generated D. suzukii line 06_F5M2 [52] by piggyBac-based germline transformation. Embryos from this line were then tested by whole mount in situ hybridization for expression of tTA, which revealed the respective cellularizationspecific expression pattern of Ds_sry α (Fig.…”

“…Since direct expression of effector molecules potentially causing harm obstructs the generation of transgenic lines, we aim to establish the tet-off binary system in D. suzukii to develop transgenic improvements for SIT approaches. To examine this binary expression system, we used the Ds_β2t enhancer/ promoter [52] to generate construct HMMA389 ( Fig. 2a).…”

“…To establish φC31-based site-specific germline transformation by integration of a transgene construct into a single attP site, we injected donor plasmid HMMA182 carrying an EGFP transformation marker and the bacterial attachment sequence attB along with helper plasmid HMMA098 expressing φC31 integrase under the promoter of the Ds-hsp70 gene into pre-blastoderm embryos of the DsRed-marked transgenic embryonic driver line 06_F5M2 (attP#1). This line was generated with construct HMMA006 [52], which harbours in addition to the early embryonic tTA-driver also an attP site (Figs. 1b, 3A).…”

“…To drive the heterologous transactivator of such a binary expression system to cause effective reproductive sterility [9,10] or female-specific killing [11,13,14,49] based on early embryonic lethality, the promoter/enhancers of cellularization-specific genes need to be identified and isolated. Moreover, to direct sperm-specific expression for transgenic marking [50][51][52] or the development of multifactorial reproductive sterility [53], the use of promoters/ enhancers active during spermatogenesis are of interest.…”