Fig. 2

Intron targeting-mediated donor plasmid knockin at the zebrafish osr2 locus. A: Schematic representation of an intron-targeted mediated strategy for generating GFF knockin at zebrafish osr2 locus using the CRISPR/Cas9 system. The sequence of sgRNA target is shown in red, and the protospacer adjacent motif (PAM) sequence is depicted in green. The right and left arm sequences of the donor plasmid are denoted by the brown lines. The left arm is 840 bp, and the right arm is 840 bp. Following co-injection of the donor plasmid with the zCas9 mRNA and sgRNA, the whole donor plasmid fregment was integrated into the osr2 locus. The zebrafish osr2 has 4 exons, with E2, E3, and E4 representing the 2nd, 3rd, and 4th exons, respectively. B: PCR analysis of the 3′ and 5′ junctions of F1 progenies from the 3# founder. The F1, R1, F2 and R2 primers are shown in A. C: 5′ junction sequences of F1 progenies of four donor plasmid knockin F0 founders. The indel mutations are highlighted in red. D: Transcription schematic of osr2 in TgKI(osr2:GFF) and WT zebrafish. E: RT-PCR was used to verify the transcription of osr2 and GFF in TgKI (osr2:GFF) and WT zebrafish (n = 3 biological replications per group). F: Schematic diagram for obtaining TgKI(osr2:GFF) homozygotes, TgKI(osr2:GFF) heterozygotes, and Tg(osr2:EGFP) transgenic fish. G: In vivo confocal image (dorsal view) of Tg(osr2:EGFP)embryos at 24 hpf reveals EGFP expression in the pronephros as indicated by the red arrowheads. Scale bar, 500 μm. H–K: The expression patterns of slc20a1a and EGFP were examined in 24 hpf embryos. H: WISH analysis of the expression pattern of slc20a1a in 24 hpf embryos (side view). I: In vivo confocal images (side view) of osr2:EGFP embryos at 24 hpf. Posterior gut (white arrowhead). J: WISH analysis of the expression pattern of slc20a1a in 24 hpf embryos (dorsal view). K: in vivo confocal images (dorsal view) of Tg(osr2:EGFP) embryos at 24 hpf. hom, homozygotes; het, heterozygotes; pA, poly(A). Scale bar in H to K, 500 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)