MicroRNAs regulate many developmental and physiological processes in C. elegans including ovulation, a rhythmic process that occurs every 20 minutes in adult worms. Even though microRNAs are known to be necessary for ovulation, the individual microRNAs that regulate ovulation are still largely unknown. One challenge of this work is that microRNAs can function redundantly and many individual microRNAs mutants do not display observable phenotypes. We have taken several approaches to identify individual microRNAs that function in the process of ovulation. First, we have performed a synthetic sterility screen of microRNA mutants using knockdown of a “hub gene,” hmg 1.2 RNAi. On this sensitized genetic background, we identified five microRNAs as candidates to regulate ovulation in C. elegans. These candidates are being further analyzed to characterize their role in ovulation. Second, we have analyzed microRNAs that were identified from small RNA sequencing of RNA isolated from mature oocytes. One abundant family of microRNAs in the oocyte is the miR-44 family. It is composed of four miRNAs: mir-44, mir-45, mir-61, and mir-247, that share a common seed sequence and thus are predicted to regulate shared target mRNAs. Triple mir-44 family mutants have significantly reduced ovulation rate and brood size, suggesting a role in regulating ovulation. mir-44 and mir-45 are within about 9kB of each other so double mutants were not able to be generated. Because miR-44 and miR-45 have identical mature miRNA sequences, we sought to obtain a double mutant using CRISPR. CRISPR was used to introduce a mutation in the mir-45 gene in worms carrying a deleting in mir-42/44. This mir-45 mir-42/44 double mutant is currently being analyzed to characterize ovulation defects. The goal is to identify pathways and targets regulated by specific microRNAs in the process of ovulation.