Analysis of CRISPR-SaCas9 Off-Targets In order to verify whether SaCas9 and sgRNAs led to unspecific cuts in the genome of transduced cells, we analyzed genomic DNA from DM1 edited clones, which were exposed to the action of CRISPR-SaCas9 for several weeks during cell amplification before gDNA extraction (Table S3). In particular, we focused on sgRNAs 4 and 23 and selected 20 potential off-targets having a number of mismatches between 4 and 6 (off-targets 1 to 10 for sgRNA 4 in Table S3A, and 11 to 20 for sgRNA 23 in Table S3B). Most off-target sites were located within intergenic regions or introns, except one, number 17, which was located within an exon. We PCR amplified and sequenced the corresponding off-target genomic regions from four DM1 edited clones carrying the deletion of the expanded CTG repeats (DM1-Delta clones 3, 10, 17, 22) and used a DM1 non-transduced clone (DM1), and a clone transduced only with the sgRNA4-23 lentiviral vector (DM1-sgRNA), as controls. Examination of the off-target chromatograms was followed by TIDE analysis, which should result in a cutting efficiency of ~50% if the clonal population harbors indels in one of the two alleles for the selected target. The total cutting efficiency calculated by TIDE using the non-transduced clone control sequence, ranged between 0 and 3.8% in DM1-Delta clones (see Table S3C). These values represent the signal background of each amplified genomic region and similar values were obtained also for the DM1-sgRNA clone. Therefore, we found no off-target indels in all four analyzed DM1-Delta myoblast clones, suggesting that sgRNAs 4 and 23 appeared specific in these cells.