Analysis of the transmission of the off-target mutation.

<p>(a) Detection of Cas9:sgRNA-mediated off-target cleavage <i>of Prkdc</i> OTS-4 in all founders (#1–40) by T7EN1 cleavage assay. PCR amplicon of <i>Prkdc</i> OTS-4 in all 40 founder rats were subjected to T7EN1 cleavage assay as described in methods. Total 23 founders (*) displayed cleavage bands. (b) PCR products with cleavage bands were cloned and sequenced. Sequence result showed OTS-4 indeed mutagenized in the 23 founders. Indels were also detected around 270 bp downstream of the OTS-4 in most colonies, which may be introduced by PCR amplification when Taq encountering repeat sequence. (c) Detection of Cas9:sgRNA-mediated off-target cleavage of <i>Prkdc</i> OTS-4 in 8 F1 pups derived from founder #3 by T7EN1 cleavage assay. Mutations were detected in 2 F1 pups (4 and 8). (d) DNA sequences of <i>Prkdc</i> OTS-4 in F1 pups 4 and 8. PCR amplicon of the <i>Prkdc</i> OTS-4 in founder #3-derived F1 pups 4 and 8 were sequenced. Sequencing result showed one kind of off-target mutation same as the founder #3 was detected in the offspring, indicating that off-target mutation induced by Cas9:sgRNA was heritable.