Development of conditional CRISPR systems for functional genomics and pest control of insects

• Applicant: Ying Yan, Ph.D.
• Subject Area: Animal Physiology and Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 470105316

Drosophila melanogaster is an excellent model organism for functional genomics, as a variety of genetic manipulation possibilities and genetic screening methods are established. Since a large ratio of genes produce lethal, semi-lethal or sterile phenotypes, insect strains carrying these mutations are either quickly lost or difficult to be maintained. This is also true for CRISPR-based genetic control approaches and thus strains are often not suitable for control programs relying on effective mass-rearing. This proposal suggests a novel conditional CRISPR system and a genetic control strategy named conditional CRISPR-based sterile insect technique (ccSIT). Specifically, two conditional methods via heat-induction and the Tetracycline-off (Tet-off) system will be used to regulate the expression of the RNA-guided DNA endonuclease Cas9. In addition, the potential gene targets for such a ccSIT strategy are evaluated for their mutation-associated phenotypes, including female lethality and male sterility. As a result, such ccSIT strains can be maintained as homozygotes at permissive conditions (at low temperature or tetracycline-supplemented food). Switching from permissive to restrictive conditions (elevated temperature or removing tetracycline) should eliminate all female offspring and only generate competitive but sterile males. In an SIT program, these ccSIT males could compete with wildtype males for wildtype females but produce no offspring. Since they do not pass the transgene to the wild population, ccSIT can be used as a safeguarding method to study CRISPR-based strategies. Besides serving as an innovative technique for pest control, this approach would also facilitate functional genomics that require controllable targeted mutations. The Spotted Wing Drosophila (SWD), Drosophila suzukii (Matsumura), a global pest without sustainable control methods, will be used as an insect pest model organism for the demonstration of ccSIT. In the last decade, I have worked on the genetic control of several insect species and established an excellent platform for genetic research in SWD. With the available molecular tools and SWD strains at the JLU Gießen in the laboratories of Prof. Schetelig, all our experiences in the Tet-off system and SIT, and the evaluated gene targets, we are ready to make a breakthrough in this topic and expand the toolbox for both basic and applied research in insect science.

• DFG Programme: Research Grants