Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.
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...
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Online Access: | https://doi.org/10.1371/journal.pgen.1008924 |
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doaj-4d98239c049246cda0811bc9d615a42e2021-04-21T13:53:17ZengPublic Library of Science (PLoS)PLoS Genetics1553-73901553-74042020-07-01167e100892410.1371/journal.pgen.1008924Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions.Valentine MosbachDavid ViterboStéphane Descorps-DeclèreLucie PoggiWilhelm Vaysse-ZinkhöferGuy-Franck RichardMicrosatellites 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.https://doi.org/10.1371/journal.pgen.1008924 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Valentine Mosbach David Viterbo Stéphane Descorps-Declère Lucie Poggi Wilhelm Vaysse-Zinkhöfer Guy-Franck Richard |
spellingShingle |
Valentine Mosbach David Viterbo Stéphane Descorps-Declère Lucie Poggi Wilhelm Vaysse-Zinkhöfer Guy-Franck Richard Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. PLoS Genetics |
author_facet |
Valentine Mosbach David Viterbo Stéphane Descorps-Declère Lucie Poggi Wilhelm Vaysse-Zinkhöfer Guy-Franck Richard |
author_sort |
Valentine Mosbach |
title |
Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. |
title_short |
Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. |
title_full |
Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. |
title_fullStr |
Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. |
title_full_unstemmed |
Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions. |
title_sort |
resection and repair of a cas9 double-strand break at ctg trinucleotide repeats induces local and extensive chromosomal deletions. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS Genetics |
issn |
1553-7390 1553-7404 |
publishDate |
2020-07-01 |
description |
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 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. |
url |
https://doi.org/10.1371/journal.pgen.1008924 |
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