Kim, Christine, Brock, Trisha, Hopkins, Christopher, McCormick, Kathyrn, Resch, Lauren, McBride, Kolt, Lawson, Jennifer, Saunders, Adam
[
International Worm Meeting,
2021]
Disease research is being greatly impacted by advances in genome-editing. CRISPR and its related techniques now allow the creation of precision animal models of human disease. With the ease of genetic manipulation in C. elegans, the fast lifecycle, and the wealth of phenotypic assays, they are a great genetic model, however, not all human disease variants can be inserted into C. elegans due to sequence divergence. We have developed a method of Whole Gene Humanization to overcome this drawback. First a human gene coding sequence is inserted as a gene replacement of its ortholog sequence in the C. elegans. When this rescues the abnormal function present in the locus null, the system becomes confirmed for conservation of biological function between the worm and human genes. Next, a test clinical variant is installed into the humanized locus and a set of deep phenotyping assays are performed to determine the landscape of phenotypic activity. The variant's phenotypic profile is referenced against the phenotypic profiles of training-set comprising of at least 11 strains (5 established benign, 5 established pathogenic variants, and one wt-humanized control). A receiver operator characteristic (ROC) curve is applied to the euclidean distance of the established benign and pathogenic variants from the wt-humanized control control. The harmonic mean separating benign from pathogenic, is then applied to the test clinical variant's euclidean distance from wt-humanized control. When the clinical variant shows a phenotypic cosegregation with the pathogenic that is above the harmonic mean, an assessment of abnormal function is achieved. We are applying this methodology to over 15 disease-gene targets. Functional rescue of 86% of the test disease loci was achieved which will enable disease modeling in a humanized locus.
[
International Worm Meeting,
2019]
C. elegans lifespan is shortened by mating, but must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death increases with age; surprisingly, this is not due to declining health, but to loss of protection upon self-sperm depletion. Self-sperm maintains expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of HLH-30/TFEB, a pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death, increasing the chances that mothers will survive through reproduction. The hijacking of the IIS pathway by males is combated by the mother's expression of an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize. ** If possible, I would prefer that this abstract please be considered with the abstract submitted by Lauren N. Booth, Travis J. Maures, Robin W. Yeo, and Anne Brunet ("Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes").