1017B Poster - 16. Techniques and technology
Friday April 08, 2:00 PM - 4:00 PM

Authors:
Erica Berent; Haosheng Li; Oscar Bautista; Joseph Terry; Bowen Man; Nicole Crown

Affiliation: Case Western Reserve University

Keywords:
a. microscopy; g. CRISPR/Cas9

Traditional fluorescence in situ hybridization (FISH) techniques are destructive to chromatin structure, and they are labor intensive when combined with immunofluorescence. In order to label and image specific loci without relying on FISH, we have adapted the genetically encoded CRISPR-Sirius system (Ma et al 2018) for use in the Drosophila ovary. This system consists of three components: a nuclease dead Cas9, a modified guide RNA containing MS2 stemloops, and an MCP-fluorescent protein fusion that binds the MS2 stemloops. To adapt this system into flies, we first used site directed mutagenesis to mutate the catalytic sites of Cas9 in the nanos::Cas9 vector (Port et al 2014). Second, we cloned eight copies of the MS2 step loop (Ma et al 2018) into the core guide RNA in pCFD3 (Port et al 2014) to generate pCFD3-Sirius. Lastly, we used an existing nanos::MCP-GFP fusion protein stock (BDSC#63821). To test our system, we cloned a guide RNA targeting the 359 bp repeat into our new pCFD3-Sirius vector. We then crossed the pCFD3-Sirius-359 repeat flies to nanos::GFP-MCP; nanos::dCas9 flies and immunostained the ovaries with an antibody against GFP. We will present data showing that our system is able to target repetitive sequences in the Drosophila genome and we will present our progress in targeting unique non-repetitive sequences.