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. 2014 Apr 11;3(5):362–371. doi: 10.1242/bio.20148177

Fig. 4. Analysis of off-target mutagenesis.

Fig. 4.

(A,B) Heteroduplex mobility assay (HMA) of the mutagenized off-target loci OT2-I4 (OT2-II1) (A) and OT2-II4 (B). The upper panel shows the alignment of the off-target sites with the targeting sequence no. 2. The targeting sequence of sgRNA is indicated in green box, adjacent to NGG protospacer adjacent motif (PAM) sequence in light blue box. The lower panel shows HMA of the off-target loci using genomic DNA mixtures of the 12 embryos. Multiple heteroduplex bands were shown in PCR amplicons from embryos injected with 100 ng/µL of Cas9 RNA and 25 ng/µL of sgRNA no. 2a, whereas a single band was shown from “Control” embryos without injection of the RGENs. (C,D) Subcloned sequences of off-target alterations at OT2-I4 (OT2-II1) (C) and OT2-II4 (D) were identified in the RGEN-injected embryos. Red dashes and letters indicate the identified mutations. The sgRNA targeting sequence and PAM indicate in green and light blue boxes, respectively. The size of deletions and insertions are shown to the right of each mutated sequence (Δ; deletions, +; insertions). Numbers on the right edge indicate the numbers of mutated clones identified from all analyzed clones from each genomic DNA mixture.