Artificial induction of a chromosomal double-strand break in Saccharomyces cerevisiae enhances the frequency of integration of homologous DNA fragments into the broken region by up to several orders of magnitude. The process of homologous repair can be exploited to integrate, in principle, any foreign DNA into a target site, provided the introduced DNA is flanked at both the 5' and 3' ends by sequences homologous to the region surrounding the double-strand break. I have developed tools to precisely direct double-strand breaks to chromosomal target sites with the meganuclease I-SceI and select integration events at those sites. The method is validated in two different applications. First, the introduction of site-specific single-nucleotide phosphorylation site mutations into the S. cerevisiae gene SPO12 Second, the precise chromosomal replacement of eleven S. cerevisiae proteasome genes with their human orthologs. Placing the human genes under S. cerevisiae transcriptional control allowed us to update our understanding of cross-species functional gene replacement. This experience suggests that using native promoters may be a useful general strategy for the coordinated expression of foreign genes in S. cerevisiae I provide an integrative targeting tool set that will facilitate a variety of precision genome engineering applications.

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Journal Article | Research Support, Non-U.S. Gov't

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Yellman CM

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