Advances in Aedes Mosquito Vector Control Strategies Using CRISPR/Cas9

Abstract

Advancements in genetic engineering have resulted in the development of mosquitoes with impaired vector competence, thereby limiting acquisition and transmission of pathogens. The main dengue (DENV) vector, Aedes aegypti, is an invasive species that have spread unwittingly across the world as a result of human trade and travel. The Ae. aegypti mosquito species has spread across tropical and subtropical regions, with higher presence in urban regions where rapid breeding patterns have shown in artificial containers. Identification of and treating an adequate number of mosquito breeding sites as a control measure have been done for the past couple of years, and yet improvement is far from the expectations, even with well-funded and well-organized initiatives. In order to stop the pathogen transmission, genetically modified mosquitoes (GMM) needs to be created and released. Despite many Aedes-related achievements, GMM creation has been challenging. The spread of particular genetic elements that impair vector competence, trigger deleterious recessive mutations, or skew a population's sex ratio can be used to prevent the spread of vector disease, or eradicate invasive organisms in a species-specific and eco-friendly manner. In recent years, genome editing strategies have evolved to make use of a variety of nucleases, ranging from sequence-specific zinc finger nucleases to modular TALENs (transcription activator-like effector nucleases) and most recently, RNA-guided nucleases adapted from bacterial adaptive immune systems, dubbed CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated systems). By combining these methods, a new era in gene editing had emerged. Generally, both of these gene editing technologies utilize sequence-specific nucleases to generate double-stranded DNA breaks (or nicks) in the target sequence, resulting in desired DNA modifications using endogenous DNA repair mechanisms. Since cells with DNA lesions are unable to divide further, the nuclease-generated strand breaks must be rapidly repaired by the cell to maintain the viability. CRISPR/Cas has been widely accepted for use in a variety of organisms, including insect species, with only minor optimization steps needed thus far. CRISPR/Cas9 technology transformed the process of engineering nucleases capable of cleaving complex genomic sequences. A complementary guide RNA (gRNA) directs the Cas9 endonuclease's operation to the specific DNA target site, enabling the editing of virtually any DNA sequence without complex protein engineering and selection procedures. Apart from genome editing, the specificity and flexibility of the CRISPR/Cas9 method enables unprecedented rapid development of genetically modified organisms with mutation systems for disease vector insect control. The stability and expression of the gene construct generated by CRISPR/Cas9 or any other method must be addressed before GMM are released, in order to make sure that pathogen transmission and formulation are interrupted robustly and completely. Spreading foreign antipathogen genes through gene drive strategies among wild mosquito populations strengthens the case for a more streamlined approach. Major fields that must be adequately assessed include risk evaluation and management, conducting studies to ensure human and environmental protection, developing effective control strategies built on comprehensive gene-driving systems, and adequately addressing the ethical, legal, and social consequences of GMM release. Although GMM is theoretically feasible as a disease control method, field releases should be made only when strong scientific evidence of human and environmental protection and effectiveness are presented, and public acceptance is addressed appropriately. This chapter discusses the diverse technological advances in generating Ae. aegypti mosquitoes which are resistant to dengue virus (DENV) and other diseases, as well as the biosafety and risk assessment of these procedures. Additionally, the chapter outlines a convincing path forward for developing successful genetic-based DENV control strategies based on CRISPR/Cas9, which could be expanded to control other arboviruses while maintaining biosafety.