The primary objective of this project is to provide the research community with severe loss-of-function mutations of all C. elegans genes. To date we, and the community, have identified such mutations for over14,000 of the 20,000 genes in the worm (G3 2012 2:1415-25; Genome Res. 2013 23:1749-63). Strains harboring these mutations are available through the Caenorhabditis Genetics Center and all data pertaining to the mutations is available at WormBase.
We are using three strategies to generate and isolate mutations in the remaining 6,000 genes. In the first strategy we will continue to isolate deletion mutants using targeted PCR after UV/TMP mutagenesis. This has been a major method for both nodes to identify mutations in non-essential and essential genes until recently, but will now be performed only at the Tokyo node.
In the second strategy we are using random mutagenesis coupled to Whole Genome Sequencing (WGS) to identify new mutations, focusing on mutations that occur in the remaining 6,000 genes that do not have null alleles (Vancouver node). This strategy will allow us to identify mutations in both non-essential and essential genes and to recover the mutant animals. Using WGS we have recently identified nonsense and splicing defects in over 2,000 genes, of which about 500 are among the remaining 6,000. We are currently working to isolate each of these mutations as homozygous lines, or as balanced recessive lethal strains. [Also see poster from the Mitani group on UV/TMP/WGS to identify deletions].
In the third strategy we are using CRISPR/Cas9 technology to target specific genes as a complement to the random approach described above. We are particularly interested in using this method to target the remaining members of large multi-gene families, for example kinases and transcription factors. We will also use it to expedite community requests if we do not already have hits through the deletion and WGS projects.
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