New deletion alleles for Caenorhabditis elegans Hedgehog pathway-related genes wrt-6 and wrt-10

We have generated new putative null alleles of two members of the C. elegans Hedgehog-related pathway: wrt-6 and wrt-10. The warthog (wrt) family member wrt-6 encodes a predicted secreted signalling molecule that contains an N-terminal Wart domain and a C-terminal autoprocessing domain (Hint or Hog domain) (Bürglin 1996; Aspöck et al. 1999; Bürglin, 2008). wrt-10 encodes another member of this Wrt family that contains the Wart domain but lacks the Hog domain (Bürglin 1996; Aspöck et al. 1999). To generate deletion alleles, we used CRISPR-Cas9 to target the first exon of wrt-6 or wrt-10 (wrt-6 sgRNA: 5′-GATGCTGTTACACCTCGTGT-3′, wrt-10 sgRNA: 5′-GCGGATTCCATTCATGATGG-3′). We isolated the wrt-6 (aus41) allele that contains a 49 bp out-of-frame deletion, predicted to cause a premature stop codon after 7 amino acids (Figure 1A). We also generated two wrt-10 deletion alleles: aus36 and aus37. The aus36 allele contains a 5 bp deletion, predicted to cause a premature stop codon after 33 amino acids (Figure 1B). The aus37 allele contains a 2 bp deletion, predicted to cause a premature stop codon after 34 amino acids (Figure 1B). As all these alleles are predicted to cause premature stop codons in the first exon of each gene, they are likely to represent molecular nulls. We found that these alleles are viable and have no obvious gross morphological phenotype. Therefore, in-depth phenotypic examination is required to dissect their functional role. To conclude, we have isolated specific deletion alleles for wrt-6 and wrt-10 and will therefore help reveal new insights into the functions of Hedgehog-related signaling in C. elegans. &nbsp