Additional file 1: Table S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb

Biochemical characterization of the medaka (<i>Oryzias latipes</i>) orthologue for mammalian tissue-type transglutaminase (TG2)

<p>Transglutaminase is an enzyme family responsible for post-translational modification such as protein cross-linking and the attachment of primary amine and/or deamidation of glutamine-residue in proteins. Medaka (<i>Oryzias latipes</i>), a recently established model fish, has similar functional proteins to those characterized in mammals. Previously, we found the apparent orthologues that correspond to human transglutaminases in medaka. In this study, regarding the medaka orthologue of human tissue-type transglutaminase (OlTGT), recombinant protein was expressed in an active form in bacteria cultured at low temperature. Using the recombinant protein, we biochemically characterized the enzymatic activity and also obtained a monoclonal antibody that specifically recognized OlTGT. Immunochemical analysis revealed that OlTGT was not expressed ubiquitously, unlike its mammalian orthologue, but in primarily limited tissues such as the eye, brain, spinal cord, and gas gland.</p

Additional file 3: Figure S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

GFP expression in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP]. (A, B) Tg[pax7a-hs:GFP]. (C, D) Tg[sox5-hs:GFP]. (A, C) Dorsal views of the head region magnified. (B, D) Lateral views of the anterior trunk region magnified. The signal in the tectum is especially strong in Tg[pax7a-hs:GFP] (A). Presumable pigment cell progenitors of xanthophore and leucophore (B) and xanthophore (D) on the body surface are positive for GFP in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP], respectively. (JPEG 1442 kb

Phenotypes of male Cumaná, Quare6, and Maculatus guppies.

<p>(A,C,E) Lateral aspects of adult males taken under incident light conditions. White rectangles indicate details enlarged in (B,D,F) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g006" target="_blank">Figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g007" target="_blank">7</a>. Traits are labeled with numbers according to their appearance in the text: 1, Cumaná black and orange ornaments on the dorsal fin; 2, Cumaná blue iridescent spot; 3, Cumaná ventral black margin of the caudal peduncle; 4, Cumaná orange-black lining of the tail fin; 5, Quare6 tail fin color pattern; 6, central black spot; 7, central orange spot; 8, Quare6 posterior black spot on caudal peduncle; 9, Maculatus black spot and whitish ornaments on the dorsal fin. We investigated the ultrastructure of traits 2, 4 (orange part), 5, 6, 7, 8, and 9 (whitish part). Scale bars: (A,C,E) 0.5 cm; (B,D,F) 500 µm.</p

Ultrastructure of Cumaná blue spot.

<p>(A,B) TEM images of Cumaná blue spot. An image of the blue spot taken under incident light conditions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g002" target="_blank">Figure 2B</a> (trait 2). Dermal iridophores and hypodermal iridophores and melanophores contribute to the spot. The epidermis was detached during sample preparation in (A). D, dermis; H, hypodermis; MU, muscle; S, scale; SP, stratum spongiosum of dermis. For other abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g003" target="_blank">Figure 3</a>. Individual from which image (A) was taken was post-fixed with osmium tetroxide. Scale bars: 5 µm.</p

Ultrastructure of fin color patterns.

<p>(A,D,F) Detail images of regions boxed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g002" target="_blank">Figure 2A, 2C, and 2E</a> taken under incident light conditions. (D) and (F) are from the individual shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g002" target="_blank">Figure 2E and 2C</a>, respectively. (A) is from a Cumaná male different from the one shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g002" target="_blank">Figure 2A</a>. (B,C,E,G) TEM images. (A) Cumaná orange-black margin of tail fin (trait 4). The ultrastructure of the orange part is shown in (B) and (C). (D) Maculatus dorsal fin ornaments (trait 9). The ultrastructure of the whitish part is shown in (E). (F) Quare6 tail fin pattern (trait 5). The ultrastructure of the whitish area is shown in (G). For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g004" target="_blank">4</a>. Individual from which image (C) was taken was post-fixed with osmium tetroxide. Scale bars: (A,D,F) 500 µm; (B) 5 µm; (C,G) 1 µm; (E) 2 µm.</p

Pigment cell types of the guppy.

<p>Xanthophores (X), melanophores (M) and iridophores (I) on the dorsal side of a guppy female shown under incident light. Leucophores could not be identified. Scale bar: 200 µm.</p

Ultrastructure of Cumaná central orange spot.

<p>(A,B) TEM images of Cumaná central orange spot. An overview image of the pigment cell distribution is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone.0085647.s001" target="_blank">Figure S1</a>. An image of the central orange spot taken under incident light conditions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g002" target="_blank">Figure 2B</a> (trait 7). Dermal xanthophores and hypodermal xanthophores and iridophores contribute to the spot. For abbreviations see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g003" target="_blank">Figures 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085647#pone-0085647-g004" target="_blank">4</a>. Individuals from which images were taken were post-fixed with osmium tetroxide. Scale bars: (A) 2 µm; (B) 1 µm.</p

Additional file 1: Table S1. of Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb

TEM images of guppy chromatophore types.

<p>(A) Melanophore on top of a scale in the dermis. Melanophores can be recognized by their dark-appearing pigment organelles, the eumelanin-containing melanosomes. (B) Melanophores, xanthophores, and iridophores in the hypodermis of the central orange spot of a Maculatus male. (C,D) Dermal xanthophores within the central orange spot of a Cumaná male. BL, basal lamina demarcating the boundary between the epidermis and dermis; E, epidermis; EO, external osseous layer of scale; I, iridophore; IF, internal fibrillary plate of scale; M, melanophore; MR, microridges of the epidermis; N, nucleus; SC, stratum compactum of dermis; ve, small vesicles or granules described in the text; X, xanthophore; xa, xanthosomes. Asterisks exemplarily mark artifacts caused by sample preparation; inflated empty spaces within iridophores are not marked. Individuals from which images (B-D) were taken were post-fixed with osmium tetroxide. Scale bars: 2 µm.</p