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

Abstract

SummaryMicrosatellites 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 theStreptococcus pyogenesCas9 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 onRAD52,DNL4andSAE2, 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 arad50Δ strain, whereas they were impaired in asae2Δ 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 thatSpCas9 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.