Au, Vinci, Moerman, Donald, Rougvie, Ann, Edgley, Mark, Doell, Claudia, Vargas, Marcus, Palma, Wilber, Hutter, Harald, Park, Heenam, Knott, Julie, Sternberg, Paul, Fernando, Lisa, Juciute, Viktorija, Li-Leger, Erica, Flibotte, Stephane, Martin, Kiana
[
International Worm Meeting,
2021]
We are continuing our community resource project to generate gene deletions of high value to those interested in human biology and disease. Our highly coordinated three-site knockout (KO) production strategy has branches in California, Minnesota, and British Columbia, with the Canadian branch transitioning from the University of British Columbia to Simon Fraser University upon the retirement of KO legend Don Moerman. We employ a dual-pipeline strategy to generate KOs, with the Minnesota and Canadian sites using a scheme based on the Calarco Lab's dual selection cassette method. It deletes all or most of the target gene, effectively eliminating function. In addition, it replaces the gene with a fluorescent reporter, allowing simple tracking of the allele in cases where the mutation results in homozygous lethality or sterility, or fails to cause a discernible phenotype, facilitating genetic manipulation of the alleles. Alternatively, the California site developed and uses a "STOP-IN" cassette that places stop codons in all three reading frames near the beginning of the coding region, eliminating activity. This method does not allow visual tracking of alleles but has the advantage in that it inserts a unique guide RNA recognition site, enabling reversion of the locus to wild-type allowing the phenotype to be reassessed. This feature is desired by some users, for example those performing metabolomics experiments requiring quantitation of phenotypes that are sensitive to strain background effects. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and promptly deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information. To date, 1,040 of our KOs are available through the CGC. Going forward, we plan to target an additional 2500 C. elegans orthologs of human genes, prioritizing known or suspected human disease genes, druggable gene classes, as well as understudied genes that are conserved to humans but that have no actionable information. Finally, we will consider community requests to prioritize specific genes.