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

Valentine Mosbach, David Viterbo, Stéphane Descorps-Declère, Lucie Poggi, Wilhelm Vaysse-Zinkhöfer, Guy-Franck Richard
doi: https://doi.org/10.1101/782268
Valentine Mosbach
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
5Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR7104 CNRS/Unistra, INSERM U1258, 1, rue Laurent Fries BP 10142, 67404 Illkirch, France
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David Viterbo
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
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Stéphane Descorps-Declère
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
4Institut Pasteur, Center of Bioinformatics, Biostatistics and Integrative Biology (C3BI), F-75015 Paris, France
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Lucie Poggi
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
2Sorbonne Universités, Collège doctoral, F-75005 Paris, France
3Biologics Research, Sanofi R&D, 13 Quai Jules Guesde, 94403 Vitry sur Seine, France
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Wilhelm Vaysse-Zinkhöfer
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
2Sorbonne Universités, Collège doctoral, F-75005 Paris, France
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Guy-Franck Richard
1Institut Pasteur, CNRS, UMR3525, 25 rue du Dr Roux, F-75015 Paris, France
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  • For correspondence: gfrichar@pasteur.fr
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Summary

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.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted September 25, 2019.
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Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements
Valentine Mosbach, David Viterbo, Stéphane Descorps-Declère, Lucie Poggi, Wilhelm Vaysse-Zinkhöfer, Guy-Franck Richard
bioRxiv 782268; doi: https://doi.org/10.1101/782268
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Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal rearrangements
Valentine Mosbach, David Viterbo, Stéphane Descorps-Declère, Lucie Poggi, Wilhelm Vaysse-Zinkhöfer, Guy-Franck Richard
bioRxiv 782268; doi: https://doi.org/10.1101/782268

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