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. 2017 Nov 23;7:16174. doi: 10.1038/s41598-017-16279-4

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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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Allele-specific cleavage of L527R TGFBI mutation utilising a PAM-specific approach. (a) The L527R mutation (c.1580 T > G) is indicated in red and PAM utilised is shown in green. A 20 nt sgRNA targeted to a naturally occurring PAM was designed as a positive control (sgWT, purple –top of figure). A 20 nt sgRNA utilising the novel PAM, containing the L527R mutation, was designed (sgMUTANT, blue – bottom of figure). (b) Both sgWT and sgMUTANT were targeted to a luciferase reporter plasmid containing either a wild-type or mutant TGFBI sequence to determine potency and allele specificity. (c) An in vitro digestion with Cas9 protein complexed with a sgRNA utilising the novel L527R PAM was carried out to confirm the specificity observed. Mutant guides of both 20 and 18 nucleotides were tested. Uncropped gel images are available in Supplementary Figure 1.