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. 2019 Jul 23;9:10645. doi: 10.1038/s41598-019-46378-3

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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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Characterization of L-ARKO zebrafish. (A–D) Images of zebrafish larvae after whole mount in situ hybridization. 4 dpf L-ARKO larvae were subjected to whole mount in situ hybridization against the ceruloplasmin probe (A), cas9 antisense probe (B) and cas9 sense probe (C). 4 dpf WT larvae was examined against cas9 antisense probe (D). (E) PCR examination of cas9 DNA in the liver, tail fin and heart of the F2 L-ARKO zebrafish. WT liver was used as a negative control. (F) T7E1 mutagenesis assay for the CRISPR target site in the ar gene in the WT zebrafish liver and F2 L-ARKO zebrafish liver, tail fin and heart. both WT and mutant ar bands are indicated with arrowheads. (G) Sequences of ar gRNA target sites. The target sites were amplified by PCR from liver DNA of an F2 L-ARKO fish and sequenced. The gRNA targeted sequence is underlined and the mutations in red. The type of mutation and frequency in percentage are indicated. All of the mutated sequences caused reading frame changes and premature termination codons.