Small RNA interactions with transgenes in genetically modified mosquito lines

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Project Summary: Strategies to use genetically modified mosquitoes for applications to control vector-borne disease rely on the activity of a synthetic transgene that can encode new phenotypes, such as mosquito-killing or virus blocking phenotypes. Observations that transgenes can unexpectedly lose functionality is consistent with regulation of the foreign DNA by an RNA interference mechanism called the piRNA pathway. Preliminary small RNA sequencing in transgenic line of both Aedes aegypti and Anopheles stephensi with such loss of function phenotypes has revealed targeting of transgenes by siRNAs and piRNAs to different degrees. The goal of this study is to identify potential small RNA activity against transgenes in mosquitoes and to directly test whether such small RNA can repress transgene activity. The first aim is to assay for siRNAs and piRNAs by enrichment and small RNA sequencing in the mosquito stages and dissected tissues that are relevant to transgene activity and inheritance in both Ae. aegypti and An. stephensi wild-type and transgenic lines and to integrate that data along with published transgene sequences into a bioinformatics resource that will support hypothesis testing in other labs and contexts. A major output of this will be a resource that can allow researchers to design constructs to avoid small RNA recognition. The second aim is to directly test whether siRNAs and piRNAs detected in transgenic lines are repressive; we will do this using a transgenic sensor strain in each species background, Ae. aegypti and An. stephensi. The strain will contain a genomic copy of with a fluorescent marker designed to be detected by specific small RNAs. If small RNAs are repressive, the offspring of a cross between the sensor strain and the test strain will result in decreased fluorescence. If siRNA and piRNAs are responsible for this phenotype, knockdown of siRNA and piRNA biogenesis proteins DICER2 or Zucchini respectively will rescue fluorescent protein expression. Activity from this cross will also be assayed by testing the abundance of reporter transcripts as well as small RNAs produced against the reporter by sequencing, which can provide information on whether repression is at the transcriptional or post-transcriptional levels. Broadly, this study will begin to identify the breadth and impact of small RNA targeting in existing transgenic lines and lay the foundation to explore how RNA interference in wild mosquitoes will respond to implementation of transgenic technologies to control mosquito-borne disease.