Evaluation of genetic engineering and genome editing tools to develop multifactorial reproductive sterility or killing sperm systems for the improvement of the Sterile Insect Technique

Abstract

Insects fulfill fundamental roles for the preservation of ecosystems such as pollination, but also represent key agricultural pests and human disease vectors accounting for immense economic losses and approximately one million deaths annually. While conventional strategies often entail environmentally damaging side effects and have failed to provide sustainable solutions to control insect pest and vector populations, the Sterile Insect Technique (SIT) has proven to be a powerful, species-specific and therefore environmentally sound tool in insect pest management. The principle of SIT is based on periodic inundative mass releases of male insects sterilized by ionizing irradiation, which results in infertile mating and ultimately in the decimation of the population. In order to mitigate the adverse effects of radiation on male fitness and competitiveness in classical SIT, and to enable efficient sex separation for male only releases, as well as to facilitate reliable monitoring by distinctive marking, several transgenic approaches have been devised and established in a variety of pest and vector species over the past twenty years. In addition, the use of engineered site-specific homing-based gene drives for insect pest control is currently heavily discussed. Successful and efficient germline transformation remains a major obstacle and laborious task that aggravates the development of new and the transfer of existing transgenic SIT approaches in non-model pest and vector organisms. Therefore, we demonstrated, to the contrary of a previous publication, that employing helper plasmids encoding for a recently engineered hyperactive version of the most commonly used piggyBac transposase significantly enhances germline transformation rates in three different species of two different insect orders. Moreover, I present my advances in the bioengineering of novel “killing-sperm” transgenic sterilization systems that could help to replace radiation in causing reproductive sterility. In a first approach, I started to bioengineer a killed-sperm system in the medfly Ceratitis capitata as an alternative approach to induce male sterility. However, the attempt to specifically kill the sperm has so far not been successful and needs further improvement on the time of expression or the use of genes causing apoptosis. In a second approach, I provide a perspective on using CRISPR/Cas in transgenic SIT to induce multifactorial sterility, which should be less sensitive to resistance development and therefore similar to irradiation-based approaches but specific to the sperm. In a third approach, I started to bioengineer a novel and innovative killer-sperm-based reproductive sterility system, in which males transfer along with their sperm a lethal factor that kills receiving females. Such a system should greatly improve SIT effectivity, as it not only guarantees male sterility but also restrains females from polyandrous mating and oviposition or blood sucking activities. Furthermore, in respect to the use of site-specific homing-based gene drives for insect pest control, we generated a Cas9-based homing gene-drive element causing a female to male sex conversion in D. melanogaster and showed that non-homologous end joining increased the rate of mutagenesis at the target site. This resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive, which indicates that simple homing CRISPR/Cas9 gene-drive designs will be ineffective.