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. 2017 Aug 21;7:8993. doi: 10.1038/s41598-017-08356-5

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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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Direct fusion of GFP-coding sequence into a chromosomal locus. (a) Schematic showing integration of the GFP-coding fragment into the Sed1 CDS. The CRISPR-Cas9 plasmid targeted (indicated by lightning bolts) the end of the signal peptide-coding region of Sed1. Dashed boxes indicate homologous regions. Left arm is homologous to the end of to the signal peptide-coding sequence (bp 8 to 57 with respect to the start of the Sed1 coding sequence). Right arm is homologous to Sed1 sequence (bp 58 to 107 with respect to the start of the Sed1 coding sequence) with substitution at bp 84 (G to A; indicated by lower-case letter in red font) to eliminate a PAM. (b) The successful integration rate was calculated as PCR check * 100. (c) Representative microscopic image of positive transformants (Nej1°/GFP_Sed1) under bright-field (left) and fluorescent (right) illumination. Insets show 3-fold magnified images.