CRISPR/Cas9 knockout of microtubule-severing katanin complex function in C. elegans neurons

HUB 302-75

Microtubule cutting by the katanin complex is important in neurons at sites of branch formation, in the process of neural pruning, and in the repair of microtubule defects. In C. elegans neurons, katanin may also be involved in axon growth and maintenance. The katanin complex is composed of a catalytic subunit and a regulatory subunit. Null mutations in the gene encoding the catalytic subunit cause embryonic lethality. The genes F47G4.4 and F47G4.5 encode regulatory subunits of katanin expressed in neurons. Worms with null alleles for these genes, ok2219 and ok2667 respectively, do not have an apparent external phenotype, but there may be abnormalities when neurons of single mutant animals are observed with fluorescent markers. Strains containing fluorescent markers targeted to different parts of the neuron were mated to strains with the ok2219 and ok2667 alleles. The mutant strains will be observed using fluorescence microscopy and compared to wildtype worms. If the genes are redundant, knocking out both genes in the same animal may provide the only true elimination of the function of the katanin complex’s regulatory subunit in neurons. To generate a double mutant, the CRISPR/Cas9 system is being used to knock out F47G4.5 in the F47G4.4(ok2219) strain. Modified methods from Norris et al. and Dickinson et al. were used to make necessary constructs to perform the CRISPR/Cas9 knockout. The overall phenotype and neuron morphology of the resulting double mutant strain may provide insight into the function of the katanin regulatory subunits.