Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

摘要

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting singlestrand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for genesilencing in mosquitoes. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracicinjection, and Cas13a transcripts were detectable at least 10 days post-delivery. The target specificcrRNA was synthesized in vitro using T7 RNA polymerase. The Cas13a plasmid and target crRNAcan be delivered by intrathoracic injection together, or the Cas13a construct can be provided first,and then target crRNA can be given later when appropriate. The machinery was tested in twomosquito species. In Anopheles gambiae, vitellogenin gene was silenced by Cas13a/Vg-crRNA, whichwas accompanied by a significant reduction in egg production. In Aedes aegypti, the α- and δ-subunitsof COPI genes were silenced by Cas13a/crRNA, which resulted in mortality and fragile midguts,reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable whena cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcriptswere observed in the study. In addition to dsRNA or siRNA mediated RNA interference, theprogrammable CRISPR interference method offers an alternative to knock down genes inmosquitoes.