#Summary
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
#Strains
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
#Experimental Design
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.