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. 2017 Jun 8;7:3036. doi: 10.1038/s41598-017-02785-y

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Optimization of the mutation/cutting efficiency at the pROSA26 locus in PFFs. (A) Scheme for site-specific targeting/cutting of the pROSA26 locus on chromosome 13 by CRISPR/Cas9. (B) Chromatograms of the PCR amplicons for each CRISPR/Cas9 dose group (0 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg and 50 μg). The red arrow indicates the cleavage site, and the PAM is underlined in red. The mutation efficiency for each CRISPR/Cas9 dose (5 µg, 10 µg, 20 μg and 30 µg) was determined by using the T7E1 cleavage assay. M: 100 bp DNA ladder. NC: Negative control, transfection dose of CRISPR/Cas9 was 0 µg. The blue arrows indicated wild type bands and the red arrows indicated cleaved amplicons. (C) The TA cloning and sanger sequences were analysed for indels. The wild-type sequence is located on the first line (WT), and the mutated sequences from the TA cloning are arranged below (T1~T12). The target site is indicated in green; the PAM is indicated in red.