Investigating the Role of Genes Y53F4B.9 and C01G12.9 in C. elegans Fertility Defects

• Grace Aitken, Biological Sciences, University of Delaware

• Amber Krauchunas, Biological Sciences, University of Delaware

Understanding genes critical for fertility enhances knowledge of reproductive biology and gamete function. This project focused on two main goals: (1) testing whether Y53F4B.9 causes the spe-3 phenotype and (2) investigating whether the C01G12.9 gene is essential for fertility. The spe-3 mutant is a loss-of-gene-function responsible for the dead egg phenotype in C. elegans. To test the first hypothesis, Y53F4B.9 was PCR-amplified from wild-type worms, purified and injected into wild-type worms, and a line carrying a GFP-tagged (Green Fluorescent Protein) extrachromosomal array was generated, allowing for gene expression specifically in the body wall muscle. After crossing this array line into a spe-3 mutant background, we observed 25% of F2 GFP- progeny to be homozygous for the spe-3 phenotype, as expected for the negative control. Conversely, 100 % of the F2 GFP+ worms produced viable progeny, indicating rescue of the sterile phenotype, which reveals that Y53F4B.9 is the causative gene of spe-3. To investigate the role of C01G12.9, CRISPR-Cas9 genome editing was performed to introduce an NheI restriction site containing a premature stop codon in the coding sequence. PCR genotyping was then performed to eventually screen the worms with an Nhe1 insert in the C01G12.9 gene. Despite multiple rounds of optimization, including testing on wild-type worms, different reagents, annealing temperatures, and two new primer sets, no PCR products were observed, suggesting unresolved difficulties. My next step is to use primers that have been previously validated to amplify the entire C01G12.9 gene. The successful rescue of spe-3 by Y53F4B.9 provides evidence for its identity and that its function is critical for the production of viable progeny in C. elegans. Continued efforts on C01G12.9 aim to clarify its role and expand our understanding of fertility-related genes in C. elegans.