Developing Genome Editing Tools for Functional Genetics in the Major Malaria Vector, Anopheles funestus

Quinn, Charlotte (2024) Developing Genome Editing Tools for Functional Genetics in the Major Malaria Vector, Anopheles funestus, Thesis (Doctoral), Liverpool School of Tropical Medicine.

Text C Quinn_00266286_Thesis_Submission_Final.pdf - Accepted Version Restricted to Repository staff only until 27 November 2025. Download (9MB)

Abstract

Malaria remains a deadly global health threat, resulting in over 600,000 deaths in 2022. While insecticide-based interventions have been most effective against disease vectors, the emergence of insecticide resistance (IR) jeopardizes this progress. It is therefore imperative to extend the efficacy of current tools and develop new control methods.

CRISPR genome editing offers insights into many aspects of mosquito biology relevant to their efficiency as vectors of disease including IR and furthers the possibility of novel genetic control approaches such as gene drives. However, for Anopheles funestus – a significant and
increasingly dominant malaria vector – the development of functional genetics tools has lagged. This thesis opens up functional genetics in An. funestus and explores two applications of CRISPR genome editing to examine putative IR alleles and genetic control via gene drives.

Firstly, Chapter 2 outlines an optimised transformation system based on the germline delivery of CRISPR components that allows efficient cleavage of a previously validated genomic site (white), and preferential repair of these cut sites via HDR to introduce defined exogenous template sequence, rather than end-joining repair. The high rates of HDR observed opens the possibility homing-based genetic control, as well as introducing defined mutations of interest without the need of a marker gene.

Based on the high rates of HDR observed, Chapter 3 describes a CRISPR-mediated delivery and screening system used to first introduce a point mutation in the genome that has been implicated in IR (‘resistance-to-dieldrin’ or Rdl296S), and then recover edited mosquitoes by non-destructive PCR. The Rdl296 allele was then functionally validated as an IR-conferring SNP using WHO tube bioassays, showing that organisms containing the allele (on an otherwise susceptible background) have an increased resistance to dieldrin (AOR = 6.5), in addition to showing that Rdl296S confers low fitness costs in terms of larval development time and adult longevity.

Thirdly, Chapter 4 outlines the design of a homing gene drive (HGD) targeting the An. funestus doublesex (AfdsxF) locus. Here it is shown that disrupting the AfdsxF locus causes an intersex-like morphology and infertility in females homozygous for the disruption, while males
and heterozygous females are largely unaffected. This mimics the effects exhibited by the same mutation in An. gambiae. A HGD construct was then inserted to show that homing is observed at relatively high rates in the male. Unexpectedly, however, females are sterile. Although in need of refining, these preliminary results overall augur well for the prospect of self-sustaining genetic control targeting population suppression in An. funestus.

In summary, this thesis demonstrates the successful use of CRISPR-HDR to broaden the study of functional genetics in An. funestus. Results generated here are anticipated to inform proven insecticide-based vector control strategies as well as novel strategies such as gene drives in the species.

Item Type:	Thesis (Doctoral)
Subjects:	QU Biochemistry > Genetics > QU 460 Genomics. Proteomics QX Parasitology > QX 20 Research (General) QX Parasitology > Insects. Other Parasites > QX 510 Mosquitoes QX Parasitology > Insects. Other Parasites > QX 650 Insect vectors
Faculty: Department:	Biological Sciences > Vector Biology Department
Depositing User:	Lynn Roberts-Maloney
Date Deposited:	27 Nov 2024 09:28
Last Modified:	03 Dec 2024 11:06
URI:	https://archive.lstmed.ac.uk/id/eprint/25697

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