Generation of an endogenous auxin inducible degron-tagged SPAS-1/spastin to investigate its targeted depletion in C. elegans neurons.

Abstract

To facilitate investigations of the microtubule severing protein spastin and its specific role in neurons, we aimed to create a C. elegans strain in which the spastin homolog SPAS-1 is visible and can be degraded with spatial and temporal precision. We used CRISPR-Cas9 to fuse an auxin-inducible degron and mScarlet to the endogenous SPAS-1 protein, enabling degradation of SPAS-1 in neurons during desired life stages. DNA sequencing confirmed in-frame insertion with the SPAS-1 N-terminus and fluorescence microscopy revealed endogenous SPAS-1 throughout the CRISPR-edited worms. Auxin treatment in rgef-1::TIR1; mScarlet::AID*::3xFLAG::spas-1 animals reduced mScarlet::SPAS-1 fluorescence in neuronal ganglia.