Developmental timing of epidermal seam cells in Caenorhabditis elegans

Abstract

Much is now known about how microRNAs downregulate gene expression by binding to sequences in the 3’ untranslated region of messenger RNAs. Less is understood about the regulation of miRNA gene expression. In this study, we are identifying the gene regulatory elements that temporally and spatially control mir-241 expression in the nematode Caenorhabditis elegans. mir-241 encodes a microRNA that times the development of epidermal “seam” cells. In absence of mir-241 expression epidermal seam cell fates can be inappropriately timed. A consequence of this is that affected adult seam cells fail to make alae, a cuticular ridge that runs the lateral length of the animal and is easily scored. Here, we are using genome editing technology to engineer deletions in the endogenous mir-241 gene regulatory region and monitor their phenotypic consequence on epidermal seam cell fate timing. The CRISPR-Cas9 system is used to generate double-stranded breaks in a region previously suspected to contain a mir-241 epidermal seam cell enhancer sequence, and randomly sized deletions are identified following repair of these breaks by the non-homologous end joining machinery. To date, a seam cell regulatory element has been narrowed to 149 base pairs. Within this region, we have identified a candidate repeated sequence that could serve as a transcription factor binding site. Current approaches are focused on generating smaller, more precise base pair deletions using a donor template for homology directed repair. Specifically, a homology donor template was designed to delete 23 base pairs that contain the candidate repeated sequence.