Development of CRISPR-Interference (CRISPR-I) for sequence-specific repression of gene expression in Aedes aegypti

CRISPR interference (CRISPR-I) combines a catalytically inactive Cas9 protein (dCas9) with repressors of transcription and a single guide RNA (sgRNA) for targeted silencing of transcription. CRISPR-I silencing differs from RNA interference (RNAi) in that CRISPR-I can be used to target specific tissues and life-stages. However, all CRISPR-I systems developed to date utilize repressor domains not conserved in invertebrates. To develop a CRISPR-I system in Aedes aegypti, we first sought to develop a reporter system that could activate a fluorescent protein upon the inactivation of a target gene. To accomplish this, we targeted the immune response pathway in insects. We used orthologs of transcriptional repressors in Aedes aegypti based on a high throughput identification of human transcriptional effectors. To test these repressors, we transfected Aedes aegypti cell lines with the dCas9 fused to the repressors and used microscopy to measure the fluorescence protein from the reporter plasmid to quantify the amount of repression. Aedes aegypti, tissue and life-stage specific repression can be achieved which has a multitude of applications in vector biology for genetic pest management use in modification of wild mosquito populations.