Expanding the toolbox for genetic modification in medfly

Siddall, Alexandra (2023) Expanding the toolbox for genetic modification in medfly. Doctoral thesis, University of East Anglia.

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Ceratitis capitata, commonly known as the Mediterranean Fruit Fly or medfly, is an invasive Tephritid pest with a wide geographic spread and potential to expand further due to climate change. With the ability to damage over 300 species of fruit and vegetable crops, this agricultural pest is of major economic importance. Control of medfly has previously relied upon pesticides and Sterile Insect Technique (SIT). With the advent of CRIPSR/Cas9 new and improved methods of genetic pest control are possible. To develop new technologies such as gene drives (drivers of a transgene through inheritance bias) and improve existing ones, the toolbox for genetic modification in medfly must be expanded. Here I develop and validate several endogenous, germline specific Cas9 promotors, one of which was subsequently used for the development of the first homing drive (drives which utilise homology-directed repair) in medfly. The sex determination pathway in medfly provides several potential targets for sex conversion, including the conserved genes transformer (tra) and doublesex (dsx) and the masculinising signal of Maleness-on-the-Y (MoY). Guide cassettes were designed to target both tra and dsx, with two tra guide constructs being synthesised and used to generate transgenic lines. Crossing these with Cas9 lines gave no evidence of cutting, and subsequent investigations revealed an error in the guide designs, prompting a redesign of the guides for future use. The use of MoY to induce masculinisation is another promising method of sex conversion in medfly, though the lethal effects of overexpression of MoY need to be overcome. A construct was designed to package MoY into gfp-tagged sperm cells by placing it under the control of the spermatogenesis specific promotor of s2-tubulin. This aimed to create a transgenic line that could induce masculinisation through MoY-carrying sperm and reduce the temporal expression of MoY to a smaller window to avoid lethality. The results revealed post-microinjection embryonic death, indicating overexpression of MoY was causing lethality. A further Cre-Lox construct was designed for future usage which should allow for the transgenic line to be established without lethality during microinjection. The final part of the genetic toolbox focused on essential genes. Potential essential genes were identified and sequenced across two wildtype, laboratory-maintained populations to check for sequence conservation. Based on these results, guides targeting these genes were designed, alongside rescue versions of these genes the guides should be unable to target. In this research, several important toolbox parts were designed, synthesised and tested in medfly which can contribute to the future development of genetic pest control strategies.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 26 Jun 2024 14:51
Last Modified: 26 Jun 2024 14:51
URI: https://ueaeprints.uea.ac.uk/id/eprint/95696


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