Project Summary/Abstract The objective of this project is to extend the C. elegans genetic toolkit available to researchers by providing publicly available gene knockout (KO) mutations in genes of high value to those interested in human biology and disease. C. elegans is a proven model for discovery of gene function and for embedding genes into functional pathways, many of which were discovered in this transparent animal and are conserved in humans. Loss-of-function mutations are the gold-standard for genetic analysis and allow inferences of gene function and interaction. A relatively recent trend is that large-scale screens (e.g., RNA-seq, proteomics, natural variation studies) generate long candidate gene lists for researchers to sort through and functionally validate, requiring loss of function mutants in many genes to be examined. Having a community-driven resource generate KOs in such genes provides reproducibility and an economy of scale that benefits all. We have developed an efficient and high throughput CRISPR-Cas9 based gene knockout (KO) project to provide a set of precisely edited gene knockout strains to the communities of C. elegans researchers and human geneticists. We seek to continue the production phase of this project, thereby transformatively increasing the efficiency and impact of C. elegans molecular genetics. We employ a highly coordinated three-site production strategy to generate KOs. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information for distribution to the community. Strains are advertised through both the CGC and WormBase websites. We have generated over 1,000 KOs to date; these KOs have supported a variety of research projects – spanning from mechanistic studies of human variants to metabolomics – funded by at least 14 NIH Institutes and Centers, demonstrating the high impact of our resource. We propose to continue to use our established pipelines to target an additional 2500 C. elegans orthologs of human genes. We will prioritize 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. This is a multi-PI project which includes the lead-PIs of the CGC and of WormBase and a subcontractor who maintains multiple community-centered databases, including a CRISPR guide RNA selection site.

Project Narrative The goal of this renewal project is to use a high throughput CRISPR-Cas9-based pipeline for the systematic generation of gene knockout mutations in the model organism, C. elegans. Targets will be selected to be of high value to human geneticists and others interested in human biology and disease. Availability of these mutations will promote research in new areas of high importance to human health, in part by attracting additional scientists from these areas to explore this premier model organism.