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. 2019 Dec 27;9:19902. doi: 10.1038/s41598-019-55681-y

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© The Author(s) 2019

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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Guide RNA design and in vitro cleavage analysis. (A) Rice phytoene desaturase gene (OsPDS) on chromosome 3. Dark green boxes represent exons, light green box and box arrow represents 5′ and 3′ untranslated regions, respectively. Two gRNA target sequences (in exon 1) are in blue and PAM sequences are in red. (B) In vitro cleavage assay for two target sequences PDS1 and PDS2. (C) In vitro cleavage assay for top two off-target sequences with highest homologies for PDS1 and PDS2. 2 m, 2 minutes; 30 m, 30 minutes; 3 h, 3 hours. a = target plasmid alone, b = target plasmid + gRNA 1 or 2, c = target plasmid + SpCas9, L = 1 kb DNA ladder.