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 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.
Author summary With the discovery of highly specific DNA endonucleases such as TALEN and CRISPR-Cas systems, gene editing has become an attractive approach to address genetic disorders. Myotonic dystrophy type 1 (Steinert disease) is due to a large expansion of a CTG trinucleotide repeat in the DMPK gene. At the present time, despite numerous therapeutic attempts, this dramatic neurodegenerative disorder still has no cure. In the present work, we tried to use the Cas9 endonuclease to induce a double-strand break within the expanded CTG repeat of the DMPK gene integrated in the yeast genome. Surprisingly, this break induced chromosomal deletions around the repeat tract. These deletions were local and involved non-homologous joining of the two DNA ends, or more extensive involving homologous recombination between repeated elements upstream and downstream the break. Using yeast genetics, we investigated the genetic requirements for these deletions and found that the triplet repeat tract altered the capacity of the repair machinery to faithfully repair the double-strand break. These results have implications for future gene therapy approaches in human patients.