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. 2017 Nov 29;7:16554. doi: 10.1038/s41598-017-16328-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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CRISPR/Cas9 mediated HDR strategies using OVL ssODN templates. (a) Schematic illustration of the rat Ephx2 locus with the site of CRISPRs/Cas9 cleavage (arrow) and the 3 overlapping ssODN (OVL1-OVL2-OVL3) of concordant polarity used as DNA repair template. The 3 overlapping ssODN are designed to generate a 228 nt insert at the rEphx2 locus. Each ssODN had a 120 nt length. The two external ssODN (OVL1 and OVL3) had a 33 nt overlap with the internal ssODN (OVL2) and a 33 nt HA corresponding to the left or the right side of the DSB. (b) Schematic illustration of the rat Ephx2 locus with the site of CRISPRs/Cas9 action (arrow) and the 3 overlapping ssODN (OVL1-OVL2-OVL3) of discordant polarity used as DNA repair template. The 3 overlapping ssODN are designed to generate a 228 nt insert at the rEphx2 locus. Each ssODN had a 120 nt length. The two external ssODN (OVL1 and OVL3) had a 33 nt overlap with the internal ssODN (OVL2) and a 33 nt HA corresponding to the left or the right side of the DSB. (c) Sequencing of the subcloned PCR products from embryos for which partial OVL ssODN insertion have been observed. 5′HA and 3′HA are underlined. Deletions are indicated in bold by “Δ”, insertions by “Ins” and in bold uppercase. OVL1, 2 or 3 insertions are indicated respectively in green, blue and red.