CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.

Abstract

The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul-1) and gRNAs (each at 100 ng ul-1) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.