To confirm that the nickase treatment specifically targeted the extreme DM1 repeat expansion only, well-known stable, non-pathogenic CNG-rich repeats in AR, ATN1, ATXN1, PPP2R2B, TBP, and TCF4 were screened for off-target activity by PCR amplification and Sanger sequencing and aligned against the background of the iDM2900 cell line (Table S4). The gene discussed is ATN1; the disease is myotonic dystrophy type 1.