C. elegans is a premier model organism that has proven highly useful for discovery of gene function and embedding genes into functional pathways, many of which were discovered in this transparent animal and are conserved in humans. In addition, other nematodes are crucial parasites of humans, infecting roughly a third of the world's population and plant-parasitic nematodes are recognized as one of the greatest threats to crops throughout the world. Despite these extensive ties to human health and disease, nematode specific genes are vastly understudied. We will use the latest CRISPR technology to knockout genes and provide a set of high value genetic tools to the communities of C. elegans researchers, human geneticists, and parasitic nematologists. Targets chosen will be 1000 C. elegans orthologs of genes implicated in human disease as well as 500 conserved genes about which essentially nothing is known, and 500 nematode-specific genes present in human parasites. We will develop an efficient pipeline of gene disruption that includes target choice, oligonucleotide design and ordering, molecular biology, microinjection into worms, selection or screening of conversion events, homozygosing or balancing alleles and verification. Edited strains will be grossly phenotyped and deposited in the CGC for distribution and advertised though the CGC and WormBase websites. We will begin by evaluating two approaches. One approach selects for gene conversion and disruption using a selectable insertion cassette; the other uses insertion of GFP as a zero-length translation fusion and will be screened by PCR. Over the first year we will identify and focus on the approach that is of higher throughput. We will continuously refine targeting methods and improve the pipeline to increase the range and efficiency as well as decrease the cost of production. We will develop Cas9 variants to increase the availability of editing sites within genes. We will develop a PCR bridge method to allow cloning free addition of homology arms. To efficiently screen for in frame GFP edits we will develop a low cost ELISA assay for GFP. Finally, we will also explore using the transcriptional activator Gal4 as a marker. This is a multi-PI project which includes the lead-PIs of the CGC, of WormBase and of the Knockout Consortium; two of the PIs have made important contributions to CRISPR technology development in C. elegans.
The goal of this project is to develop a new CRISPR-Cas9-based pipeline for the systematic generation of marked gene knockouts in the model organism, C. elegans. Targets will be selected to be of high value to human geneticists and parasitic nematologists, including genes implicated in human disease and genes conserved among nematode parasites of human, respectively. 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.
| Wang, Han; Park, Heenam; Liu, Jonathan et al. (2018) An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9. G3 (Bethesda) 8:3607-3616 |
| Au, Vinci; Li-Leger, Erica; Raymant, Greta et al. (2018) CRISPR/Cas9 Methodology for the Generation of Knockout Deletions in Caenorhabditis elegans. G3 (Bethesda) : |
| Schwartz, Hillel T; Sternberg, Paul W (2018) A Toolkit of Engineered Recombinational Balancers in C. elegans. Trends Genet 34:253-255 |
