DYN-1 is a GTPase protein whose function is critical for endocytosis. A previous study in C. elegans isolated a recessive temperature-sensitive dyn-1 mutation that caused worm paralysis at temperatures over 25 °C. These mutants, termed dyn-1(ky51), possessed a serine to proline substitution at amino acid 70 within the GTPase domain of the DYN-1 protein. Because DYN-1 is an essential protein, complete knockouts of the dyn-1 gene cause embryonic lethality. The dyn-1(ky51) mutation is therefore, an attractive tool to study DYN-1 function as the loss of DYN-1 activity in this mutant is reversible. However, the mechanism by which the P70S substitution perturbs DYN-1 function is presently unknown. Further, the original dyn-1(ky51) mutation was created by EMS mutagenesis. The EMS mutagenesis method is known to non-specifically introduce background mutations within the worm genome which may in turn influence the phenotypes of the dyn-1(ky51) mutation. To unambiguously investigate the effect of the dyn-1(ky51) mutation on C. elegans, we re-created this mutation in C. elegans utilizing the CRISPR/Cas-9 technology. The dyn-1(ky51) mutation was introduced into C. elegans by microinjecting worms with CRISPR/Cas9 editing reagents. The repair template for introducing the dyn-1(ky51) mutation was designed to include a PstI restriction site. Positively edited progeny of the injected parent worm were genotyped and screened by PCR followed by NdeI digestion and agarose gel electrophoresis. Upon identifying positively edited worms, the dyn-1(ky51) mutation was homozygosed by screening the progeny of the positively edited worms for the presence of homozygous edits. Thus, we were able to successfully create a homozygous-edited dyn-1(ky51) C. elegans worm line by CRISPR. Future studies with this mutant allele will involve elucidating the mechanism by which the dyn-1(ky51) mutation perturbs the dynamics of the DYN-1 protein using biochemical assays and the artificial intelligence molecular modeling system AlphaFold.

Erin Haastrup

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