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. 2017 Aug 7;7:7411. doi: 10.1038/s41598-017-07606-w

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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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Deletion of the ASAH1 gene in A375 melanoma cells. (A) Schematic structure of the ASAH1 gene. Red bars indicate gRNA recognition sites, arrows indicate the annealing of nested PCR primers. Predicted guide score and possible off-target sites are also shown. (B) Nested PCR analysis of the ASAH1 gene showing events ofdeletion in heterozygosis (a, b, c, e), in homozygosis (f, g, h, i). (C) Surveyor Nuclease Assay on two loci indicated in panel (A) demonstrating the lack of detectable off-target activities of the guides utilized in the present study. (D) RT-PCR analysis shows lack of ASAH1 transcription in deleted clones. ASAH1 transcripts are still present in clones with single insertion/deletion (InDel) mutations (c).