Bactofection is a promising biotechnology that is gaining considerable attention as an alternative medical treatment for many human diseases. In this approach, Escherichia coli are altered with two genes that enable them to deliver RNA hairpins or plasmids to a host. The Invasin gene facilitates bacterial endocytosis so naturally extracellular bacteria become intracellular. Once intracellular, the bacteria lyse enabling their genetic contents to disperse. Listeriolysin O (LLO), which allows intracellular bacteria to exit host vesicles, is used in this system to facilitate the escape of bacterially derived RNA hairpins or plasmids from the host vesicle, where they can then modulate host gene expression or be transcribed. Here, we will adapt the principles of bactofection to mosquitoes enabling bacterial delivery RNAi and clustered regularly interspaced short palindromic repeats (CRISPRs) to these medically important insects.
In specific aim one, we will optimize the bacterial delivery system for Anopheles gambiae and manipulate mosquito gene expression using RNAi. To optimize the delivery system to insects, we will examine knock down efficiency when a short hairpin RNA (shRNA) is expressed from a bacterial or mosquito promoter in addition to examining various mutated forms of the LLO protein, which have been shown to increase bactofection in mammalian systems. We will then use this insect optimized bactofection system to silence genes in both larvae and adult mosquitoes. CRISPRs are an exciting and ever-expanding molecular tool that has recently been modified to manipulate gene expression. This is accomplished by targeting a dead Cas9 (dCas9) protein to the promoter regions, which can either inhibit or enhance the transcription. In the second aim, we will develop CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for use in mosquitoes and use the optimized bactofection system to deliver these molecules to mosquitoes. This will enable gene specific knock up and knock down in Anopheles mosquitoes. For both shRNA and CRISPR delivery, bacteria will be administered to mosquitoes by simply inoculating the larval water or spiking their sugar meal. This technology will provide a flexible and simple system for manipulating mosquito gene expression and be a dramatic improvement on current methods. This approach is suitable for modulating gene expression in larval, pupal and adult life stage. Additionally, this system has the potential to be scaled up for genome wide screening for gene silencing and gene activation and can be used both in vitro and in vivo. In additional to being an invaluable laboratory tool, this approach has the potential for use as a novel vector control strategy.
RNAi, a method to silence gene expression, has been used for over a decade to elucidate the function of many insect genes, including genes that influence pathogen development in mosquitoes. While RNAi will continue to be a key research tool in vector biology, the use of CRISPRs to both enhance and suppress gene expression offers news opportunities for manipulating mosquito transcription. Here we will develop a new delivery method, which is a great improvement on current delivery techniques, whereby bacteria transfer RNAi and CRISPRs to mosquitoes enabling efficient manipulation of gene expression.
