Figure 7.

Cas9-induced HPRT mutagenesis. (A) Design of exon 6-targeted gRNAs. Part of the exon 6 sequence in WT cells or clone 5.2.1 is shown. The I-SceI site is boxed with non-WT residues shown in bold and the cleavage site indicated (staggered arrows). Target-specific regions of gRNAs are shown, aligned with their target. For each gRNA, the predicted Cas9 cleavage site is indicated by an arrow, 3 nt upstream of the protospacer-adjacent motif (underlined). (B) Stained petri dishes after selection of transfected HT1080 or clone 5.2.1 cells (10⁶ cells per dish) in 6TG. Cells were lipofected with DNA encoding no nuclease (column 1), Cas9 and the indicated gRNA (columns 2–5), or I-SceI (column 6). Cells from the same transfection shown in (B) were plated at lower dilutions and selected in 6TG and used to determine frequencies (Materials and Methods; Table 3, Experiment 1). (C) Relative proportions of different classes of I-SceI- or Cas9-induced mutations (data from Fig. 4D and E; Supplementary Material, Fig. S3).