The Gilleard lab at University of Calgary has identified a new beta-tubulin variant in canine hookworm that appears to confer resistance to benzimidazoles. In collaboration with the Gilleard Lab, we have created this allele, Q134H, in the ben-1 gene of a susceptible strain of Caenorhabditis elegans, N2. Edited strains were created using CRISPR-Cas9 genome editing, and two unique edits were generated to control for any potential off-target effects. Edited strains, the susceptible N2 strain, and a strain with a deletion of ben-1 were exposed to albendazole and response was measured using a previously described high-throughput assay. Results show that this allele confers equal levels of resistance as the deletion of ben-1
N2- Susceptible laboratory strain
ECA882- Resistant edited strain with deletion of ben-1
ECA3353 and ECA3358- Independent edits of Q134H in ben-1 of the N2 background
For the duration of the experiment, worms were maintained at 20 degrees Celsius. A 0.5 cubic centimeter chunk from stock plates for each strain was placed of the agar plates seeded with OP50 bacteria. Two days after, adults were spot bleached on a new plate. Briefly 10-20 gravid worms were placed into a 15 microliter spot of bleaching solution placed on on a new plate, to synchronize the C. elegans populations. L1 worms were divided onto three new plates the following day, placing 20 worms per plate. Two days after, nine plates of 5 L4 worms were picked from the L1 plates for each strain. 72 hours later, worms were bleached to collect synchronized embryos. Embryos were collected in K-media and then concentrated at a concentration of 1000 embyros/milliliter of K-Media. 50 microliters were dispensed into two rows of 11 eleven wells each of four 96 well plates. The following morning, 25 microliters of lypophilized HB101 bacterial lysate, containing albendazole, was dispensed into each well. 48 hours later, plates were treated with 5 micro-molar sodium azide and imaged on the COPAS Biosort worm sorter.