Previous forward and reverse genetic screens have identified a handful of genes that when their expression is abrogated lead to defects in the development of the somatic gonad in C. elegans. Many genes that are important for gonadal development could be genetically redundant; alternatively, they could be essential for the viability of the organism. Either case would cause the genes not to have been easily isolated in previous screens. To help identify additional genes responsible for promoting gonadal development, a cell-specific transcriptional approach was conducted that identified transcripts enriched for expression in the developing somatic gonad compared to the whole animal in each sex. We are now working to further study how the gonad-enriched transcripts are driving the development of the somatic gonad in either sex. We are using CRISPR-Cas to N-terminally tag the endogenous loci of gonad-enriched genes of interest with GFP. To generate the GFP-tagged strains, we are using a self-excising cassette (Dickinson et al. 2015). Prior to the eviction of the self-excising cassette, the self-excising cassette is inserted between the promoter and open reading, disrupting the gene expression, and allowing for us to test the loss-of-function phenotype. After excision, the GFP-tagged protein is generated for expression studies. Of the gonad-enriched transcripts, approximately 15% are encoded by essential genes, and therefore the loss-of-function gonadal phenotype has not been possible to study. To determine the role that these essential genes are playing within the gonad, we are modifying a tool that has been shown to lead to tissue-specific degradation of genes (Wang et al. 2017). We are constructing a strain that allows us to degrade GFP-tagged proteins exclusively in the gonad, allowing us to test the gonadal function of essential genes. Finally, as part of a Course-based Undergraduate Research Experience, we are using CRISPR-Cas to create serial deletions in the upstream regulatory promoter of the
fkh-6 gene, which is important for gonadal development of both sexes. We have identified a region in the regulatory promoter that leads to feminization of male gonads but does not drastically change the expression pattern of FKH-6 in either sex.