Ciliopathies are inherited genetic disorders caused by mutations in over two hundred cilia-associated genes. Whole genome or exome sequencing can identify the genetic cause of ciliopathies. When pathogenic variants are identified, this information can be used for genetic counselling, adjusting clinical care based on gene-specific complications, and even to qualify patients for gene-specific clinical trials. However, many missense variants are termed "variants of unknown significance" (VUS), which cannot be classified as pathogenic or benign based on standard in silico methods. Here, we have used the small roundworm Caenorhabditis elegans and CRISPR/Cas9 genome editing to model and characterize missense VUS alleles. Specifically, we have generated two benign, two pathogenic, and seven VUS alleles in mks-3
, the nematode orthologue of the Joubert Syndrome and Meckel-Gruber syndrome (MKS) gene TMEM67. MKS-3 is a member of the MKS protein module at the ciliary base transition zone (TZ). Employing quantitative assays of cilium structure and function, as well as protein localisation analyses, we found that the known benign alleles phenocopy wild type controls, whereas the known pathogenic mutants display severe cilia defects that are phenotypically similar to those of a reference null deletion allele. Of the seven VUS alleles, four are strongly pathogenic, two are benign, and one displays an intermediate phenotype. We conclude that C. elegans is an excellent model organism for in vivo interpretation of VUS alleles in conserved cilia genes. Such data may be beneficial for diagnosis of genetically unresolved disease, and understanding genotype-phenotype correlations in ciliopathies.