Genetic Engineering using TAL proteins in Yeast

Using model organisms is a needed intermediate step when developing potential therapeutics. In this study, yeast is used as a model organism to find out how TAL effector nuclease work inside a living cell. TALEs are a family of protein that contains a DNA binding domain which consists of a number of repeats. Each repeat is identical except at the 13^th and 14^th amino acid which is unique and codes for the nucleotide that the specific repeat binds to. Nucleases can be attached to these TALEs which can then act to both knockout genes through non-homologous end joining (NHEJ) or to add a gene sequence through homologous recombination.

To test these proteins in yeast, three gene targets were selected in yeast: URA3, LYS2, and ADE2. The first test looked at how well mutations, either deletions or insertions, could be induced through NHEJ using TALEs with nucleases attached to them. The DNA binding regions were built to recognize and bind to regions within the three genes of interest. The various mutant genotypes for each gene were used to measure the efficacy of each TALE. Most of the data showed a fairly small change in the overall number of cells from .15-5.4% of them showing some sort of mutation from a TALE nuclease. For studying homologous recombination, resistance genes were added that had homologous regions on either side matching with the targeted area in the URA3 gene. The addition of TALE nucleases revealed a fairly high degree of recombination with up to 34% of the cells having the resistance gene.

Even if these proteins could target the right segment of DNA that does not mean that is the only place they can be targeting. To test against this the different TALEs were inserted into haploid cells and grown. If the TALE nucleases are cutting at off target sites, we would expect to see decreased growth due to deleterious mutations. Yeast is a good model for looking at this, as haploid cells would show these mutations more than a diploid would. The data shown in the study show that the TALE infused cells were identical in growth to that of the control. In addition, the sequence of these cells was determined and no insertion / deletions were found other than the ones targeted for in the specific TALE tested.

So what we have here is a protein that can effectively alter DNA within cells in an efficient and specific manner. The implication of this study is that these proteins could one day be used in humans to alter genetic information in vivo. Although gene therapy is still a work in progress, TALE proteins seem to be another potential, and powerful, tool. All this thanks to your favorite model organism, Saccharomyces cerevisiae.

Li, T. et al. Modularly Assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acid Research. (2011). Dpo:1-.093/nar/gkr188.