Highly compound mutation of mir-51-56 (miR-100) microRNA binding sites is phenotypically silent in C. elegans

These mutants display developmental delay, reduced brood size, mating defects, and behavior and gene expression changes reflective of caloric restriction. To assess the functional importance of mir-51 family targets in these phenotypes, we queried a list of putative targets using a focused RNAi screen and mRNA-sequencing. Through these efforts, we narrowed down a candidate list of 31 mir-51 family targets, which we investigated for their potential to recapitulate mir-52; mir-54-56 phenotypes upon derepression.

To this end, we used CRISPR to mutate the miRNA binding sites in all 31 putative target genes, generating single mutant strains as well as multiple highly compound mutant strains with up to thirteen genes with mutated miRNA binding sites. Surprisingly, single and highly compound binding site mutants were largely wild type and do not recapitulate phenotypes of mir-52; mir-54-56. These results may suggest that the mir-51-56 family loss of functions phenotypes are driven by the simultaneous derepression of a large number of targets and thus technically challenging to effectively model genetically.