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Fig. 1. Targeted integration of egfp into krt12.2.L locus and expression of eGFP
(A) Scheme of the donor DNA integration into 5' UTR of the target gene. Light and dark blue boxes indicate 5'UTR. Brackets show homologous sequences containing promoter and 5UTR. Red bars indicate sgRNA target sequences in the target locus and the donor plasmid. CDS, coding sequence of the target gene. DSB/NHEJ, DNA double strand break and non-homologous end joining. Indel, insertion and/or deletion. FW1/RV1 and FW2/RV2 indicate primer binding sites to amplify upstream and downstream junctions, respectively. (BD) Representative images of a krt12.2.L:eGFP embryo at NF stage 45 injected with fgk537b-egfp. Clear eGFP fluorescence signal was observed in fin and gill. Signal was also found in pronephros and olfactory/oral region. (B), bright field image. (B), fluorescence image of (B). (C, D), magnified partial images of (B). g, gill. pn, pronephros. ol, olfactory pit. Asterisks indicate guts with autofluorescence. Bar in B, 1 mm. Bars in C and D, 500 m. Representative images of tadpoles injected with fgk246b-egfp and fgk66b-egfp are shown in Figure S2 (E) Expression phenotypes of injected normal embryos were classified at around NF stage 40. Specific (blue), fin-and-gill-specific expression in both sides or either side. Partial (yellow), partial expression in fin and gill. Ectopic (gray), ectopic or ubiquitous expression. Rates of classified embryos are indicated as percentages in graphs. Donor DNAs and total numbers are indicated at top and bottom of graphs, respectively. Results of four (fgk567b-egfp and fgk246b-egfp) and five (fgk66b-egfp) independent experiments are combined. (F) PCR analysis of genomic DNAs from un-injected wild-type and embryos expressing eGFP with the specific pattern. Lanes 13, 46 and 79 indicate embryos injected with fgk537b-egfp, fgk264b-egfp and fgk66b-egfp, respectively. Left and right gels show PCR fragments including upstream and downstream junctions, respectively. Primers for upstream (krt12.2.L gFW and egfp RV1) and downstream (vector FW1 and krt12.2.L gRV) are shown in Supplemental Table 3. The predicted size of the upstream PCR fragment is 1062 bp if the donor is integrated in the forward direction with no indel. Predicted sizes of the downstream fragments are 849 bp, 552 bp and 312 bp for embryos injected with fgk537b-egfp, fgk264b-egfp and fgk66b-egfp, respectively. The amplified fragments were sequenced and the sequences obtained are shown in Fig. S3.
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Fig S1. Sequence of krt12.2.L fragment in plasmid fgk537b-egfp.
Gray and yellow highlighted letters indicate upstream and 5'UTR sequences of krt12.2.L. Underline indicates sgRNA target sequence with PAM sequence (red underline). Two arrows indicate primers for cloning of krt 12.2.L 264bp and 66bp fragments (fgk264b, fgk66b).
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Fig S2. Representative images of fgk264b-egfp and fgk66b-egfp embryos.
(A, A' Bright field and fluorescence images of fgf246b-egfp-injected embryo. (B, B') Bright field and fluorescence images of fgf66b-egfp-injected embryo. g, gill. pr, pronephros. ol, olfactory pit. Bars, 1 mm.
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Fig S3. Sequences of the junction regions between the integrated donor DNAs and the targeted krt12.2.L locus.
Partial sequences of 5 UTRs, including upstream (upper) and downstream (lower) junctions, from embryos injected with fgk537b-egfp (1-3), fgk264b-egffp (4, 6) and fgk66b-egfp (7-9) are shown with the wild-type sequence. Downstream junction sequence from embryo 5 was not amplified by PCR (see Fig. 1F). Underline indicates the sgRNA target sequence with PAM sequence (red underline). Inserted nucleotides are indicated in red. Images show examples of sequence chromatograms for the junction regions. Indicated sequences are complementary to the 5UTR sequence. Blue arrows indicate the deletion sites with the deleted sequences in parenthesis.
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