TY - GEN T1 - Resolving a contradiction: full gene deletion of kqt-3 by CRISPR does not lead to pharyngeal pumping defects AU - Bauer, Rosemary AU - Nebenfuehr, Benjamin AU - Golden, Andy DO - 10.17912/MICROPUB.BIOLOGY.000137 UR - https://www.micropublication.org/journals/biology/micropub-biology-000137/ AB - KQT-3 is the C. elegans ortholog of human KCNQ1, a voltage-gated potassium channel that is implicated in multiple types of Long Q-T cardiac arrhythmias including Jervell and Lange-Nielsen syndrome and Romano-Ward syndrome. In order to assess the possibility of modeling these syndromes in C. elegans, we obtained the tm542 deletion allele, which had previously been associated with changes to the defecation cycle (Nehrke et al., 2008, Kwan et al. 2008). Any visible, easily-scored phenotype would be an indication that we could move forward with creating patient-specific missense alleles by CRISPR. The tm542 deletion, a 1001bp deletion generated by chemical mutagenesis that disrupts exons 2-4, appeared to cause a dramatic reduction in pharyngeal pump frequency (Fig 1B). This was an indication that mutations to kqt-3 might be a reasonable way to model Long Q-T arrhythmias in worms. When three patient missense mutations and two smaller deletions yielded only very subtle changes to pharyngeal pumping, we became increasingly suspicious of the tm542 deletion strain. This prompted the creation of a full-length, 9004bp deletion allele by CRISPR that starts 69bp upstream of the start codon, ends 66bp after the stop codon, and inserts stop codons in every frame. Surprisingly, complete deletion of this 621 amino acid protein led to virtually no change in pharyngeal pumping (Fig 1B-D). In order to confirm that the pharyngeal pumping phenotype in the kqt-3(tm542) strain was due to extraneous mutations and not a dominant negative effect, we created the full-length deletion of kqt-3 by CRISPR in the tm542 strain. This kqt-3(tm542av187Δ) deletion strain phenocopied the original tm542 deletion (Fig 1B-D), indicating that other mutations besides the deletion in kqt-3 are responsible for the lack of pharyngeal pumping in the tm542 strain. This study should serve as a cautionary tale for any lab studying a deletion allele that was generated by chemical mutagenesis; the strain likely contains numerous other mutations. We now routinely generate a full gene deletion by CRISPR as part of any genetic study. PY - 2019 PB - microPublication Biology ER -