Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

Abstract

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. In this study we developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes and tested the machinery in two mosquito species. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least 10 days post-delivery. In Anopheles gambiae, vitellogenin (Vg) gene was silenced by Vg-crRNA injection two hours post blood meal, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the α -and δ subunits of COPI genes were knocked down by a post-blood meal crRNA injection, which resulted in mortality and fragile midguts, reproducing a phenotype reported in a RNAi mediated COPI gene silencing study. The silencing of mulitple genes simlutaneously is achievable when mosquitoes are given a cocktail of Cas13a construct with crRNAs targeting respective genes.This study adds a programable CRISPR tool to manipulate RNA in insects.