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. 2018 Aug 13;8:12059. doi: 10.1038/s41598-018-30137-x

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© The Author(s) 2018

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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(A) Schematic illustration of the wild-type Pax6 locus. Exons (ex1 to ex3) and introns are indicated by black boxes and a black line, respectively. Dual guide RNAs were used on the Pax6 locus: namely, the two target sequences recognized by gRNA are overlined and the PAM sequences are shown in red. The position of primers (Rat Pax6-F and -R) used to identify indels around the target sequences is indicated by arrowheads. (B) Fetal offspring (Pax6-#1 to #8) at 16.5 dpc obtained after i-GONAD targeted towards the paired domain of Pax6. Various abnormal phenotypes such as deformity of the facial structure together with loss of eye cup (as exemplified by Pax6-#1, #2 and #8) are discernible. (C) Representative 16.5 dpc fetuses exhibiting various types of craniofacial structure. Fetus Pax6-#5 and -#3 exhibited normal facial structure, and was wild-type and heterozygous for Pax6, respectively. Fetus Pax6-#8 completely lacked eyes and the lateral nasal prominence, and was homozygous knockout for Pax6. (D) Genotyping of the fetuses shown in (B). Fetuses Pax6-#1 to #3 and #8 have heterozygous PCR products (*allele-1of Pax6-#1, **allele-2 of Pax6-#1, ^†allele-1of Pax6-#2, ^††allele-2 of Pax6-#2, ⁺allele-1of Pax6-#3, ⁺⁺allele-2 of Pax6-#3, ^$allele-1of Pax6-#8 and ^$$allele-2 of Pax6-#8). Fetuses Pax6-#4 to #7 have wild-type PCR products (1,489 bp). (E) Direct sequencing of PCR products derived from the animals shown in (B). Fetus Pax6-#5, which was judged as wild-type through morphological inspection (shown in B and C) and genotyping (shown in D), exhibited normal sequence for Pax6. In contrast, fetus Pax6-#1, which was judged as KO through morphological inspection (shown in B) and genotyping (shown in D), exhibited a 7 bp sequence deletion (underlined) in front of the PAM (shown in red). (F) Comparison of PAX6 amino acid sequences from fetuses derived from SD females subjected to i-GONAD-mediated induction of indels at the Pax6 locus. The control amino acid sequence of wild-type PAX6 protein is shown on the top. The amino acid sequences of the mutated PAX6 proteins (derived from Pax6-#1 to #3 and #8) are shown. Predicted consequences of the mutation on amino acid sequences are highlighted in red. Stop codons are shown as x. The green bar indicates the paired domain.