Development of next-generation gene drive technologies for Anopheles population engineering

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PROJECT SUMMARY Malaria is currently the most impactful mosquito-borne disease worldwide, sickening 228 million people and killing over 405,000 in 2018, 2/3 of which are young children — the most vulnerable demographic. Several mosquito species of the Anopheles genus can act as vectors of the parasite causing malaria, and in recent years their increasing resistance to pesticides is hampering current control methods and blunting our response to eventual disease outbreaks. Globalization is further allowing both vectors and pathogens to move freely and in certain situations to permanently establish themselves in new locations. CRISPR-based gene drive technologies for mosquito population engineering are being developed as they represent a new promising addition to our arsenal for fighting this disease. These technologies are up-and-coming, yet few issues have come up during their development. Briefly, a gene drive system based on CRISPR is composed of a Cas9 and a gRNA gene inserted in the mosquito genome at the location where the gRNA targets it. The arrangement of this genetic cassette endowed it with self-replicating properties that allow it to propagate to the same location on a wild-type chromosome. This property can be harnessed to spread within a population a beneficial trait that would help reducing disease transmission (population modification), or a deleterious trait to help reduce the mosquito population (suppression). While this process is extremely accurate, it can result in the failure of self-propagating, and the generation of small mutations at the targeted locus preventing further conversion by the gene drive. These “resistance alleles” generated during the drive process have been identified as a major hindrance to field applications of these tools. In addition, due to the deposition of active Cas9 and gRNA in the developing embryo, the mosquito biology allows an extensive production of such resistance alleles when a gene drive is inherited from a female. The long-term goal of this project is to develop powerful gene drive tools that can be used for the fast and reliable engineering of wild Anopheles populations. In order for these tools to be ready to have an impact on the malaria morbidity worldwide, the two issues described above need to be overcome. To tackle these two problems, in the three Aims of the proposed research, we will develop and optimize three parallel technologies in the fruit fly Drosophila melanogaster and subsequently apply them to the major malaria vector Anopheles stephensi.