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. 2014 Mar 5;9(3):e89413. doi: 10.1371/journal.pone.0089413

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© 2014 Ma et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(a) Detection of Cas9:sgRNA-mediated off-target cleavage of Prkdc OTS-4 in all founders (#1–40) by T7EN1 cleavage assay. PCR amplicon of Prkdc 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 Prkdc 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 Prkdc OTS-4 in F1 pups 4 and 8. PCR amplicon of the Prkdc 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.