Gene targeting at and distant from DNA breaks in yeast and human cells

Single-strand break (SSB);Targeted gene correction;Double-strand break (DSB);Yeast;Gene targeting

Stuckey, Samantha Anne ; Storici, Francesca Biology Chernoff, Yury Lobachev, Kirill Santangelo, Philip Schmidt-Krey, Ingeborg ; Storici, Francesca

University:Georgia Institute of Technology

Department:Biology

关键词: Single-strand break (SSB);    Targeted gene correction;    Double-strand break (DSB);    Yeast;    Gene targeting;   

Others  :  https://smartech.gatech.edu/bitstream/1853/51721/1/stuckey_samantha_a_201305_phd.pdf

美国|英语

来源: SMARTech Repository

【 摘 要 】

Here we developed multiple genetic systems through which genetic modifications driven by DNA breaks caused by the I-SceI nuclease can be assayed in the yeast Saccharomyces cerevisiae and in human cells. Using the delitto perfetto approach for site-directed mutagenesis in yeast, we generated isogenic strains in which we could directly compare the recombination potential of different I-SceI variants. By genetic engineering procedures, we generated constructs in human cells for testing the recombination activity of the same I-SceI variants. Both in yeast and human cells we performed gene correction experiments using oligonucleotides (oligos) following modification and/or optimization of existing gene targeting protocols and development of new ones. We demonstrated that an I-SceI nicking enzyme can stimulate recombination on the chromosome in S. cerevisiae at multiple genomic loci. We also demonstrated in yeast that an I-SceI-driven nick can activate recombination 10 kb distant from the initial site of the chromosomal lesion. Moreover we demonstrated that an I-SceI nick can stimulate recombination at the site of the nick at episomal and chromosomal loci in human cells. We showed that an I-SceI double-strand break (DSB) could trigger recombination up to 2 kb distant from the break at an episomal target locus in human cells, though the same was not observed for the nick. Overall, we demonstrated the capacity for I-SceI nick-induced recombination in yeast and human cells. Importantly, our findings reveal that the nick stimulates gene correction by oligos differently from a DSB lesion, as determined by genetic and molecular analyses in yeast and human cells. This research illustrates the promise of targeted gene correction following generation of a nick.