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

Fig. 5. Dose-dependency of off-target alterations by the RGEN.

Fig. 5.

Both the DJ-1 targeting locus and two disrupted off-target loci were analyzed using genomic DNA mixture of the 12 embryos that were injected with dilution series of sgRNA no. 2a and 100 ng/µL Cas9 RNA. (A) Heteroduplex mobility assay (HMA). Each analyzed locus and concentration of the injected sgRNA was shown in the upper side of the panel. (B,C) Subcloned sequence of the targeting locus observed in the embryos injected with either 10 ng/µL (B) or 5 ng/µL (C) of sgRNA no. 2a. (D–G) Subcloned sequence of the off-target locus OT2-I4 (OT2-II1) (D,E) or OT2-II4 (F,G) observed in the embryos injected with either 10 ng/µL (D,F) or 5 ng/µL (E,G) of sgRNA no. 2a. (B–G) 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.