Advancing CRISPR-Based Genome Editing Strategies for managing Aedes aegypti mosquito populations - PROJECT SUMMARY/ABSTRACT Several viruses such as yellow fever, Zika, Chikungunya, and dengue affect millions of people, causing over 300,000 deaths every year. These viruses are vectored through Aedes mosquito bites, transmitting the pathogens between people. The use of insecticides to kill mosquitoes or vaccines to protect people against the viruses has helped reduce the incidence of these diseases. However, these pathologies still represent a continuing public health and economic burden in disease-endemic countries. The advent of CRISPR has offered tremendous potential to solve this public health issue by providing new technologies such as the genetic sterile insect technique (pgSIT) and gene drive approaches. 1) pgSIT relies on the generation of two transgenic lines: a Cas9 strain and a line containing a set of guide-RNAs (gRNAs) targeting genes involved in male sterility and female lethality or infertility. Therefore, the combined genetic cross of the Cas9 and gRNAs lines produces only sterile male progeny. 2) CRISPR gene drives allow for engineering wild populations due to their ability to self-propagate and spread engineered traits. Gene-drive strategies to fight malaria aim to either spread beneficial cassettes to confer pathogen resistance for population modification or to propagate deleterious mutations for population suppression. These strategies have demonstrated efficiency under laboratory conditions; however, there are important issues that need addressing before these new technologies can be safely applied in the environment: 1. pgSIT requires the maintenance and crossbreeding of two parental strains to obtain sterile males, involving time-consuming logistical efforts when scaling for field purposes. 2. Cas9-based gene drives can introduce small insertions/deletions (indels) instead of replacing the wildtype allele with a copy of the gene drive element when the repair process fails. This represents one of the most significant obstacles to the field deployment of gene drives. My proposed studies aim to tackle these concerns. First, I will design a Cas12 temperature-dependent system; this strategy will only require one strain containing both Cas12a and gRNAs. It can be maintained at lower temperatures like a regular strain but produces sterile males when switched to higher temperatures (Aim 1). Additionally, I will address indel formation concerns by using the Cas12a nuclease that generates small deletions still recognizable by the gene drive element to allows its full introgression (Aim 2). Future technologies born from this project’s groundwork could reduce the economic burden of dengue (and other viruses) disease and increase our readiness for an eventual worsening of the disease incidence.
