Trinucleotide repeats contraction by double-strand break induction

Les répétitions de trinucléotides sont des séquences répétées en tandem pouvant subir, chez l'homme, de larges expansions à l'origine de nombreuses maladies génétiques. La dystrophie myotonique de type 1 (DM1) est due à l'expansion d'une répétition CTG en 3'UTR du gène DMPK. Les mécanismes d'instabilités des répétitions, peu connus, reposeraient sur leur capacité à former des structures secondaires constituant un obstacle aux mécanismes impliquant une synthèse d'ADN. Nous avons montré qu'une TALEN induisant une cassure double brin dans les répétitions CTG à l'origine de la DM1 insérées chez la levure Saccharomyces cerevisiae permettait de manière efficace et spécifique d'aboutir après réparation à leur contraction. Le mécanisme de réparation est dépendant uniquement de deux gènes, RAD50 et RAD52, suggérant la formation de structures aux extrémités de la DSB devant être retirées pour initier la réparation, suivis d'une réaction de SSA entre les répétitions aboutissant à leur contraction. L'efficacité et spécificité d'un système CRISPR-Cas9 à contracter ces répétitions chez la levure ont été comparées à la TALEN. L'induction de CRISPR-Cas9 n'aboutit pas à la contraction des répétitions mais à des réarrangements chromosomiques suggérant un manque de spécificité et un mécanisme de réparation différent de celui de la TALEN. Enfin, nous avons étudié si ces nucléases peuvent contracter ces répétitions CTG à des tailles non pathologiques dans des cellules de mammifères. L'induction de la TALEN dans des cellules de souris transgéniques DM1, puis dans des fibroblastes humains de patients DM1 montre des résultats préliminaires encourageant de contraction des répétitions.Trinucleotides repeats are a specific class of microsatellites whose large expansions are responsible for many human neurological disorders. Myotonic dystrophy type 1 (DM1) is due to an expansion of CTG repeats in the 3’UTR of DMPK gene, which can reach thousands of repeats. Molecular mechanisms leading to these large expansions are poorly understood but in vitro studies have shown the capacity of these repeats to form secondary structures, which probably interfere with mechanisms involving DNA synthesis. We shown that a TALEN used to induce double-strand break (DSB) in DM1 CTG repeats integrated in the yeast Saccharomyces cerevisiae is specific and leads to highly efficient repeat contractions after repair. Mechanism involved in TALEN-induced DSB only depends of RAD50 and RAD52 genes, suggesting the formation of secondary structures at DSB ends that need to be removed for repair initiation, followed by an intramolecular recombinaison repair such as SSA between repeats leading to their contraction. We compared the efficiency and specificity of a CRISPR-Cas9 and the TALEN to contract CTG repeats in yeast. Surprisingly, CRISPR-Cas9 induction do not lead to repeat contraction but to chromosomal rearrangement, suggesting a lack of specificity and a different repair mechanism than with the TALEN. At last, we studied whether these nucleases could contract CTG repeats to a non-pathological length in mammalian cells. Finally, TALEN induction in DM1 transgenic mice cells, and in DM1 human fibroblasts show promising repeat contractions

Trinucleotide repeat instability during double-strand break repair: from mechanisms to gene therapy

International audienceTrinucleotide repeats are a particular class of microsatellites whose large expansions are responsible for at least two dozen human neurological and developmental disorders. Slippage of the two complementary DNA strands during replication, homologous recombination or DNA repair is generally accepted as a mechanism leading to repeat length changes, creating expansions and contractions of the repeat tract. The present review focuses on recent developments on double-strand break repair involving trinucleotide repeat tracts. Experimental evidences in model organisms show that gene conversion and break-induced replication may lead to large repeat tract expansions, while frequent contractions occur either by single-strand annealing between repeat ends or by gene conversion, triggering near-complete contraction of the repeat tract. In the second part of this review, different therapeutic approaches using highly specific single- or double-strand endonucleases targeted to trinucleotide repeat loci are compared. Relative efficacies and specificities of these nucleases will be discussed, as well as their potential strengths and weaknesses for possible future gene therapy of these dramatic disorders

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

International audienceTrinucleotide repeat expansions are responsible for at least two dozen neurological disorders. Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6. Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded

TALEN-Induced Double-Strand Break Repair of CTG Trinucleotide Repeats

Summary: Trinucleotide repeat expansions involving CTG/CAG triplets are responsible for several neurodegenerative disorders, including myotonic dystrophy and Huntington’s disease. Because expansions trigger the disease, contracting repeat length could be a possible approach to gene therapy for these disorders. Here, we show that a TALEN-induced double-strand break was very efficient at contracting expanded CTG repeats in yeast. We show that RAD51, POL32, and DNL4 are dispensable for double-strand break repair within CTG repeats, the only required genes being RAD50, SAE2, and RAD52. Resection was totally abolished in the absence of RAD50 on both sides of the break, whereas it was reduced in a sae2Δ mutant on the side of the break containing the longest repeat tract, suggesting that secondary structures at double-strand break ends must be removed by the Mre11-Rad50 complex and Sae2. Following the TALEN double-strand break, single-strand annealing occurred between both sides of the repeat tract, leading to repeat contraction

Highly specific contractions of a single CAG/CTG trinucleotide repeat by TALEN in yeast.

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

A fast, sensitive and cost-effective method for nucleic acid detection using non-radioactive probes

International audienceNucleic acid detection and quantification using a labeled DNA probe is a very common molecular biology procedure. Here, we describe a new method, based on commonly used laboratory solutions, for nucleic acid hybridization and detection with digoxigenin-labeled DNA probes. The protocol described is faster, more sensitive and much cheaper than a standard protocol using commercial solutions. Comparison with a classical radioactive detection method shows that the latter exhibits less background and shows a greater linear response. Hence, the proposed protocol may be routinely performed for qualitative detection of nucleic acid, but when precise signal quantitation needs to be obtained, radioactive probe hybridization associated to phosphorimaging technology is more reliable

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ∼2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal rearrangements around the repeat tract. These rearrangements depended on RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This proved that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end.In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfer with gene conversion too. Altogether, these data show that SpCas9 is probably not a good choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome

Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions

International audienceMicrosatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome

Experimental design.

<p><b>A</b>: Plasmids pCLS9996 and pCLS16715, carrying the two TALEN arms, were transformed into <i>MAT</i>a and <i>MAT</i>alpha haploid strains, and strains were crossed to obtain diploids containing both TALEN arms. The TALEN is normally repressed on glucose medium. One copy of the active <i>SUP4</i> tRNA being insufficient to suppress the <i>ade2</i>-opal mutation, yeast cells are red <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone.0095611-Richard2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone.0095611-Richard3" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone.0095611-Richard4" target="_blank">[27]</a> (top). In the presence of galactose, the TALEN is expressed, binds CAG/CTG trinucleotide repeats and induces a double-strand break into the repeat tract. If a second copy of an active <i>SUP4</i> tRNA is generated during double-strand break repair, the <i>ade2</i>-opal mutation will be suppressed and yeast cells will now be white (bottom). <b>B</b>: Sequences recognized by both TALE DNA-binding domains and by the split-TALE. The length of the minimal spacer (18 bp) needed to induce a DSB was deduced from repeat tract lengths analyzed in surviving cells after TALEN induction (see text).</p

Karyotypes and sequencing of TALEN-induced yeast colonies.

<p><b>A</b>: Sanger sequencing of survivors. PCR fragment amplified with su3/su9 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone-0095611-g002" target="_blank">Figure 2C</a>) was sequenced using a primer (su7) located ca. 210 bp upstream of the repeat tract. Left: when only one allele was present, one unique sequence was read (upper graph, homozygous tract length). When two alleles of different lengths were present, the sequence was blurry and unreadable after the shortest of the two repeat tracts (lower graph, heterozygous tract length). The freeware 4Peaks was used to visualize sequences. Right: length distribution of alleles in homozygous tract length (green bars) and heterozygous tract length (orange bars) survivors to TALEN induction. Homozygous tract lengths are shorter on the average (mean = 7 triplets) than heterozygous tract lengths (mean = 9 triplets), this difference being very significant (Wilcoxon test, p-value = 0.0021). Note that for heterozygous alleles only the length of the shortest repeat can be precisely known, hence the statistical difference observed between the two distributions is more significant than shown. <b>B</b>: Deep sequencing of yeast genomes from yeast colonies isolated on glucose or galactose plates. Each of the 15 yeast genomes was resequenced to 700 X coverage, on the average (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone.0095611.s003" target="_blank">Table S1</a>). For each colony, the number of unique SNPs, insertions/deletions or size changes in other CAG/CTG triplet repeats of the yeast genome, are indicated. <b>C</b>: Pulse-field gel electrophoresis of red and white colonies after galactose induction. Chromosomal DNA was prepared from yeast cells embedded in agarose plugs according to standard methods <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095611#pone.0095611-Schwartz1" target="_blank">[72]</a>. Agarose plugs were loaded on 1% agarose gels (SeaKem GTG, TEBU) and electrophoresis was run on Rotaphor (Biometra) at 12°C in 0.25X TBE buffer at pH8.3, 140V, with decreasing pulse ramp 140 sec to 80 sec, and field angle 120°. Karyotypes are identical among all clones and do not show any large chromosomal rearrangement, neither on chromosome X (bearing <i>SUP4</i>) nor on any other chromosome. <b>D</b>: Two models proposing how heterozygous and homozygous tract lengths may be formed following TALEN induction (see text).</p