Abstract
Plasmodium relies on numerous agonists during its journey through the mosquito vector, and these agonists represent potent targets for transmission-blocking by either inhibiting or interfering with them pre- or post-transcriptionally. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities for the study of agonist function and for developing malaria control strategies through gene deletion to achieve complete agonist inactivation. Here we have established a modified CRISPR/Cas9 gene editing procedure for the malaria vector Anopheles gambiae, and studied the effect of inactivating the fibrinogen-related protein 1 (FREP1) gene on the mosquito’s susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants developed into adult mosquitoes that showed profound suppression of infection with both human and rodent malaria parasites at the oocyst and sporozoite stages. FREP1 inactivation, however, resulted in fitness costs including a significantly lower blood-feeding propensity, fecundity and egg hatching rate, a retarded pupation time, and reduced longevity after a blood meal.
Highlights
Malaria remains one of the most devastating infectious diseases, killing nearly half a million people annually [1]
The causative agent of malaria, Plasmodium, has to complete a complex infection cycle in the Anopheles gambiae mosquito vector in order to reach the salivary gland from where it can be transmitted to a human host
In our preliminary attempts to generate clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout null mutants we tried the method established for Aedes and Anopheles stephensi mosquitoes [11, 15] by co-injecting synthesized guide RNA (gRNA) and Cas9 protein into the embryos of A. gambiae G3 mosquitoes
Summary
Malaria remains one of the most devastating infectious diseases, killing nearly half a million people annually [1]. The malaria parasite has to complete a complex journey in the mosquito vector that involves intimate molecular interactions with the vector’s midgut, hemolymph, and salivary glands [2]. These crucial Anopheles-Plasmodium interactions represent targets for transmission blocking by inhibiting parasite agonists (or host factors) that are required for infection [3,4,5,6]. The exploration of mosquito Plasmodium agonists for the development of malaria control strategies based on parasite suppression has lagged behind other approaches because of the lack of effective gene editing tools for Anopheles mosquitoes. The recently developed CRISPR/Cas9-based genome editing methodology and gene-drive systems for Anopheles mosquitoes provide new and promising tools for the study of Plasmodium host factors, with regard to their biology and potential for the development of transmission-blocking strategies
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