Presentation description

E3 ubiquitin ligases are critical components of the proteasome degradation pathway, and essential for vertebrate development, yet the functions of many E3s remain unknown. Our lab uses the zebrafish (Danio rerio) as a model for examining the effects of E3 mutagenesis on morphological changes in early development. Zebrafish has proven to be an exceedingly adaptable model for genetic studies due to its high fecundity, rapid growth, and amenability to genome editing and high-throughput screening techniques. The Peterson lab has employed the zebrafish model to develop MIC-Drop (Multiplexed Intermixed CRISPR Droplets), a high-throughput CRISPR mutagenesis platform that allows CRISPR screening in a vertebrate organism. In this study, we are using MIC-Drop to mutagenize all 634 known E3s in the zebrafish, followed by morphological evaluation to identify E3s with functions in early development. Specifically, this process consists of using microfluidics to create nanoliter-sized droplets containing Cas9, gene-specific guide RNAs, and a unique DNA barcode associated with each E3 gene. The droplets are then injected into single-cell embryos-one droplet per embryo. A visual screen is conducted to identify larvae with early developmental changes, and the barcodes from these larvae sequenced to reveal the phenotype-associated E3. Our analysis and screen found that mutagenesis of vps18 caused loss of pigmentation in the eyes and skin, and hepatomegaly when compared to wild-type zebrafish. Our lab will continue to use MIC-Drop to screen for other phenotypes related to E3 loss.