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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: Nat Biotechnol. 2014 Dec 16;33(2):187–197. doi: 10.1038/nbt.3117

Figure 6. GUIDE-Seq profiles of RGNs directed by tru-gRNAs.

Figure 6

(a) Numbers of previously known and novel off-target cleavage sites identified for RGNs directed to the to VEGFA site 1, VEGFA site 3, and EMX1 target sites by matched full-length gRNAs and truncated gRNAs. Note that the data for the RGNs directed by full-length gRNAs are the same as those presented in Fig. 1e and is shown again here for ease of comparison.

(b)-(d) Chromosome ideograms showing on- and off-target sites for RGNs directed to the VEGFA site 1, VEGFA site 3, and EMX1 target sites by matched full-length gRNAs and truncated gRNAs. Note that the ideograms for the RGNs directed by full-length gRNAs are the same as those presented in Fig. 1h and Supplementary Fig. 3 and are shown again here for ease of comparison.

(e) GUIDE-Seq-based identification of DSBs induced by RGNs directed by tru-gRNAs. Mapped reads for the on-target sites of the three RGNs directed by tru-gRNAs we assessed by GUIDE-Seq are shown. In all cases, the target site sequence is shown with the 20 bp protospacer sequence to the left and the PAM sequence to the right on the x-axis. As with RGNs directed by full-length gRNAs, note how the highest peak falls within 3 to 4 bps of the 5′-edge of the NGG PAM sequence, the expected position of an RGN cleavage event.

(f)-(h) Sequences of off-target sites identified by GUIDE-Seq for RGNs directed by tru-gRNAs. For each RGN, the intended target sequence is shown in the top line with cleaved sites shown underneath and with mismatches to the on-target site shown and highlighted in color. GUIDE-Seq sequencing read counts are shown to the right of each site. The intended on-target site is marked with a green square, previously known off-target sites of RGNs directed by both a full-length gRNA and a tru-gRNA are marked with a red diamond, and previously known off-target sites found only with RGNs directed by a tru-gRNA are marked with a yellow diamond. Previously known off-target sites were those that were shown to have a mutagenesis frequency of 0.1% or higher in an earlier report27. Data is shown for RGNs directed by tru-gRNAs to the (f) VEGFA site 1, (g) VEGFA site 3, and (h) EMX1 target sites.