Tools
Tools
Purcell, Jessica (2021) Genetic Control of Mosquitoes: A modular approach to improving transgene design for complex CRISPR population control strategies, Thesis (Doctoral), Liverpool School of Tropical Medicine.
|
Text
J Purcell PhD - Thesis final.pdf - Accepted Version Download (4MB) | Preview |
Abstract
Mosquito borne diseases present an ongoing public health burden in many parts of the world. There are several, successful strategies for reducing disease burden (often by targeting the vector population) but there remains a need for additional strategies that can improve efficacy, reduce cost and reduce undesired side effects (such as the ecological impact of spraying chemical insecticides). One such group of strategies are those using genetic modification of the mosquito to control the wild vector population and reduce disease transmission. Such strategies have been increasing in complexity in recent years with the wide availability of programmable gene editing through the CRISPR/Cas system. Even as genetic control systems are developed and tested, there remains a need for optimisation in the design and function of different elements within the transgenes they are based on.
This thesis presents practical tools for the field of mosquito transgenics, particularly in the design and implementation of complex, CRISPR gene drive strategies. Using cell culture as a model, this work describes the validation of several, specific components of transgene design. The cell culture format was exploited to test a large number of variants of each component, more than would be practical in vivo. These findings are presented as an empirical resource for aid in design of mosquito genetic modification constructs.
Alternative translation initiation sequences (TIS) were characterised as a mode of modulating expression efficiency of transgenic proteins such as toxic effectors or fluorescent markers. TIS and 3’UTR sequences were identified that can be used independently or in tandem to induce 2 – 20 fold increases in transgene expression.
A CRISPRa assay was then developed and used in a pipeline for identification and validation of alternative U6 and 7SK RNA polymerase III promoter sequences to drive guide RNA expression in mosquito species of interest. Availability of multiple, sequence-divergent promoters increases capacity to express multiple guide RNAs within a single transgenic individual, a requisite for may complex CRISPR/Cas vector control strategies. This work expands on the state of the field through examining the use of exogenous promoters from closely related species. It was then demonstrated that truncations of the U6 promoters retain considerable activity, offering a route to reduce transgene cassette size.
| Item Type: | Thesis (Doctoral) |
|---|---|
| Subjects: | QU Biochemistry > Genetics > QU 470 Genetic structures QU Biochemistry > Genetics > QU 550 Genetic techniques. PCR. Chromosome mapping QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes QX Parasitology > Insects. Other Parasites > QX 600 Insect control. Tick control QX Parasitology > Insects. Other Parasites > QX 650 Insect vectors |
| Faculty: Department: | Biological Sciences > Vector Biology Department |
| Depositing User: | Lynn Roberts-Maloney |
| Date Deposited: | 22 Jun 2022 13:51 |
| Last Modified: | 22 Sep 2022 01:02 |
| URI: | https://archive.lstmed.ac.uk/id/eprint/20635 |
Statistics
Actions (login required)
 |
Edit Item |