Figure 7: Evaluation of CRISPR/Cas9 toxicity and off-target effects for deletion of human exon 51.

(a) Results of a cytotoxicity assay in HEK293T cells treated with human-optimized SpCas9 and the indicated sgRNA constructs. Cytotoxicity is based on survival of GFP-positive cells that are co-transfected with the indicated nuclease. I-SceI is a well-characterized non-toxic meganuclease and GZF3 is a known toxic zinc finger nuclease. n=3 independent transfections (mean+s.e.m.). (b) Surveyor analysis at off-target sites in sorted hDMD cells treated with expression cassettes encoding SpCas9 and the indicated sgRNAs. These three off-target sites tested in hDMD cells were identified from a panel of 50 predicted sites tested in HEK293T cells (Supplementary Fig. 6 and Supplementary Table 2). ND, not determined; OT, off-target locus; TGT, on-target locus for indicated sgRNA. (c,d) End-point nested PCR to detect chromosomal translocations in (c) HEK293T cells treated with Cas9 and CR1 or (d) sorted hDMD cells treated with Cas9, CR1 and CR5. The schematic depicts the relative location of nested primer pairs customized for each translocation event. The expected size of each band was estimated based on the primer size and the location of the predicted sgRNA cut site at each locus. Asterisks indicate bands detected at the expected size. The identities of the bands in c were verified by Sanger sequencing from each end (Supplementary Fig. 11). A representative chromatogram for the P2/P5 translocation in HEK293T cells is shown.