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. 2017 Dec 19;7:17771. doi: 10.1038/s41598-017-17651-0

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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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PCR characterisation of HDR events in CHO.S18. LPV sequences in RMCE master clone CHO.S18 were targeted by a co-transfection of DV_HDR and a Cas9/sgRNA expression vector. (a) Flow cytometry plot of the cell pool after transfection. The red rectangle indicates the gate used for single cell sorting for clonal isolation of GFP⁻/RFP⁺ cells. (b) Recombination events in 12 of the resulting clones were further characterised by PCR. In the first PCR analysis (upper panel; primers P-HDR-1 and P-HDR-2, see suppl. Fig. S9 for details) the PCR product of 1,596 bp is indicative for successful HDR events. In the second PCR analysis (lower panel; primers P-HDR-1 and P-HDR-3, see suppl. Fig. S9 for details) the entire targeted segment is amplified. Successful homologous recombination events result in an approximately 3,702 bp PCR product, whereas parental loci as well as NHEJ indels will result in a 2,825 bp PCR product. Irregular recombination events (red stars) could be detected in two cases. (P: parental clone CHO.S18). The uncropped gel pictures are shown in suppl. Fig. S11d.