Abstract
Trinucleotide repeat expansions are responsible for more than two dozens severe neurological disorders in humans. A double-strand break between two short CAG/CTG trinucleotide repeats was formerly shown to induce a high frequency of repeat contractions in yeast. Here, using a dedicated TALEN, we show that induction of a double-strand break into a CAG/CTG trinucleotide repeat in heterozygous yeast diploid cells results in gene conversion of the repeat tract with near 100% efficacy, deleting the repeat tract. Induction of the same TALEN in homozygous yeast diploids leads to contractions of both repeats to a final length of 3–13 triplets, with 100% efficacy in cells that survived the double-strand breaks. Whole-genome sequencing of surviving yeast cells shows that the TALEN does not increase mutation rate. No other CAG/CTG repeat of the yeast genome showed any length alteration or mutation. No large genomic rearrangement such as aneuploidy, segmental duplication or translocation was detected. It is the first demonstration that induction of a TALEN in an eukaryotic cell leads to shortening of trinucleotide repeat tracts to lengths below pathological thresholds in humans, with 100% efficacy and very high specificity.
Highlights
Trinucleotide repeat expansions are involved in at least two dozens dramatic neurological and developmental disorders in human [1,2,3,4,5]
White colonies were scored and represent a majority of cells on both media, even though they are more frequent on galactose (82.5% of white colonies) as compared to glucose (66.7%). This suggests that even in repressing conditions, the GAL1-10 promoter shows some level of leakiness which is, associated to multicopy plasmids, apparently sufficient to induce TALEN expression. In support of this observation, we noticed that when crossing two haploids strains containing a stable trinucleotide repeat and one of the two TALEN arms, none of the diploids obtained contained a repeat longer than 30 triplets, strongly suggesting than even in repressing conditions, leaky expression of both TALEN arms occur to a level high enough to induce repeat contractions when both plasmids are in the same diploid cell (Figure S1)
We show that a TALEN designed to recognize and cut a CAG/CTG trinucleotide repeat integrated in a yeast chromosome was 100% efficient in shortening the repeat tract, without inducing any other mutation in the yeast genome
Summary
Trinucleotide repeat expansions are involved in at least two dozens dramatic neurological and developmental disorders in human [1,2,3,4,5]. When an I-Sce I recognition site was inserted between two short (CAG) repeats and a double-strand break (DSB) was induced, two-thirds of the repair events led to shortening of the repeat array by single-strand annealing, even though a homologous template was available to repair the break by gene conversion [26]. This observation led us to the idea that inducing a specific DSB within a given trinucleotide repeat could lead to its shortening to nonpathological length
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