George Dimopoulos, Yuemei Dong, Eric Marois, Maria L. Simões, W. Harry Feinstone Department of Molecular Microbiology and Immunology [Baltimore, MD, États-Unis] (MMI), Johns Hopkins Bloomberg School of Public Health [Baltimore], Johns Hopkins University (JHU)-Johns Hopkins University (JHU), Modèles Insectes de l'Immunité Innée (M3I), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the National Institutes of Health / National Institute of Allergy and Infectious Disease grants R21AI131574 (to GD) and RO1AI122743 (to GD), and a Johns Hopkins Malaria Research Institute Postdoctoral Fellowship (to MLS), the Bloomberg Philanthropies (to GD). CNRS, Inserm, the University of Strasbourg and Agence Nationale de la Recherche (grants JCJC 'GEMM' and #ANR-11-EQPX-0022) to EM., ANR-11-JSV3-0001,GEMM,Outils génétiques pour la manipulation du génome d'Anopheles gambiae(2011), ANR-11-EQPX-0022,I2MC,Insectarium pour l'Infectiologie Moléculaire et Cellulaire(2011), Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Bodescot, Myriam, Jeunes Chercheuses et Jeunes Chercheurs - Outils génétiques pour la manipulation du génome d'Anopheles gambiae - - GEMM2011 - ANR-11-JSV3-0001 - JCJC - VALID, and Equipements d'excellence - Insectarium pour l'Infectiologie Moléculaire et Cellulaire - - I2MC2011 - ANR-11-EQPX-0022 - EQPX - VALID
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., Author summary 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. The parasite’s development in the mosquito relies on numerous host factors (agonists), and their inhibition or inactivation can thereby result in suppression of infection and consequently malaria transmission. The recently developed CRISPR/Cas9-based genome editing tools for Anopheles mosquitoes provide new and promising opportunities to delete (inactivate) Plasmodium agonists to better understand their function and for blocking malaria transmission. Here we have established a modified CRISPR/Cas9 genome editing technique for malaria vector A. gambiae mosquitoes. Through this approach we have inactivated the fibrinogen-related protein 1 (FREP1) gene, via CRISPR/Cas9 genome editing, and the impact of this manipulation on the mosquito’s susceptibility to Plasmodium and on mosquito fitness. FREP1 knockout mutants showed a profound suppression of infection with both human and rodent malaria parasites, while it also resulted in fitness costs: a significantly lower blood-feeding propensity, fecundity and egg hatching rate, and a retarded larval development and pupation time, and reduced longevity after a blood meal.