Open AccessResection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions
Author(s) -
Valentine Mosbach,
David Viterbo,
Stéphane Descorps-Declère,
Lucie Poggi,
Wilhelm Vaysse-Zinkhöfer,
GuyFranck Richard
Publication year - 2020
Publication title -
plos genetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.587
H-Index - 233
eISSN - 1553-7404
pISSN - 1553-7390
DOI - 10.1371/journal.pgen.1008924
Subject(s) - biology , trinucleotide repeat expansion , rad50 , genetics , myotonic dystrophy , homologous recombination , gene conversion , direct repeat , polyglutamine tract , cas9 , tandem repeat , genome , non homologous end joining , genome editing , dna repair , gene , mutant , dna binding protein , allele , huntingtin , transcription factor
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 sae2 Δ 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 Sp Cas9 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.