CRISPR-based innovative genetic tools for control of Anopheles gambiae mosquitoes

Abstract

Malaria and other mosquito-borne diseases pose an immense burden on mankind. Since the turn of the century, control campaigns have relied on the use of insecticide-impregnated bed nets and indoor residual sprays to stop Anopheles mosquitoes from transmitting the malaria parasite. Although these are our best strategies to control the spread of disease, wild mosquito populations are developing resistance to insecticides at an alarming rate, making disease control increasingly challenging. In the search for new powerful strategies aimed at controlling malaria-transmitting Anopheles populations, we can now exploit a suite of powerful genome engineering tools to control wild populations and monitor releases. In this dissertation we utilize CRISPR/Cas9 technology and other genetic engineering tools in Anopheles gambiae to generate genetically sterile males for population suppression, to assess the feasibility of developing evolutionarily stable gene drives for population replacement, and to expand the genetic toolkit for field releases of genetically modified mosquitoes. We develop CRISPR/Cas in A. gambiae for male genetic sterilization and for basic biological study. Using this system we generate a line of mutant mosquitoes with deletions in Zero Population Growth (ZPG), a gene critical for germ cell development. Resulting male mutants show no sperm in the testes and sterilize the females with which they mate, demonstrating that similar systems could be adapted for use in Sterile-Insect Technique (SIT)-like release campaigns. We also test whether CRISPR/Cas9 can facilitate the sustainable and stable spread of gene drives in natural mosquito populations. Specifically, we design gene drives that have the potential of being evolutionarily stable by insertion into haplolethal ribosomal genes. To facilitate this goal, we create gene drive docking lines via a novel knockin technology for insertion of complex DNA templates into genetically intractable loci. We identify multiple challenges associated with such systems, including the occurrence of Minute-like mutant phenotypes that present severe fitness costs when targeting haploinsufficient genes, a general decay in gRNA function over time that has consequences for all gene drives designed to date, and the critical need for precisely controlling Cas9 expression to avoid large fitness costs. Finally, we develop and validate a novel transgenic tool for monitoring GM field releases. We generate transgenic lines expressing a fusion of a fluorescent marker with a male seminal protein specifically in male reproductive tissues. Incorporation of this fluorescence fusion into the mating plug, which is transferred to the females during mating, allows the visual identification of successful mating events for a few hours after copulation. This transgenic tool enables effective monitoring of GM male mating competitiveness in field trials, overcoming current limitations. The work outlined here significantly expands the genetic toolkit for the manipulation of Anopheles mosquitoes, facilitating the implementation of genetic control strategies aimed at malaria-transmitting vector populations.