Genome-wide TILLING: From gene-based to organism-based screens

Targeting Induced Local Lesions IN Genomes (TILLING) or target-selected mutagenesis is a versatile and universal method for generating defined genetic mutants and knockouts. The approach is based on chemical mutagenesis to introduce random mutations in genomes, followed by large-scale targeted identification of induced mutations. This method has been shown to work very effectively in a wide variety of plants and animals. For C.elegans, we established a clonal library of 6,144 EMS-mutagenized worms. High-throughput screening by dideoxy resequencing resulted in the identification of 1,044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation or our results indicates that nonsense mutations for over 90% of all C. elegans genes are present in the library. Currently, the effectiveness of the target-selected mutagenesis method is limited by the screening capacity and as a consequence, libraries consisting of thousands of mutant samples are screened on a gene-by-gene basis. To maximally benefit from and to identify all knockouts in mutant resources novel experimental approaches need to be developed. I will discuss our efforts to further optimize and scale the target-selected mutagenesis approach towards genomewide organism-based identification of gene knockouts in C.elegans, zebrafish, and the rat. Implementation of emerging next-generation sequencing technologies and alternative approaches will be presented. (author)