Myotonic dystrophy type 1 (DM1 or Steinert disease) is a monogenic neurodegenerative disorder due to the expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene on chromosome 19. We developed a strategy to induce a DNA double-strand break (DSB) within the expanded repeat in order to contract it below the pathological range in humans. This approach was successful in yeast and mouse cells and we are now trying to apply it to human cells from a patient affected by DM1. This patient was diagnosed with a large CTG repeat expansion (≈ 2000 triplets) and we estimated the size of the repeat tract by Southern blot to be 1980 triplets. These cells (ASA cells) were immortalized by overexpression of the telomerase catalytic unit, theoretically allowing to grow them for 100 generations (instead of ca. 20 generations for primary cells).
A Cas9 nuclease directed to the expanded CTG repeat was expressed in ASA cells for several weeks and the population was subcloned. Trinucleotide repeat length was analyzed in many clones, by Southern blotting. This showed that the repeat was partially contracted in some clones, the others showing no size change. Ten clones were chosen to be sequenced by PacBio, in order to look for possible off-target mutations generated by the Cas9 nuclease.
Note that this project is a follow-up of project B3245, in which ten ASA clones expressing a TALEN had been completely sequenced using the PacBio technology. The present project aims at comparing results obtained with the TALEN to those obtained with Cas9, in order to determine which one of the two nucleases is the most specific (i.e. triggers less off-target mutations).
