Figure 2—figure supplement 2.

(A) The position and sequence of the kctd10 intron 1 (I1) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Targeting efficiency evaluated by PCR and Hpy188I restriction endonuclease digestion. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Schematic diagram of the kctd10-2A-td GFP floxP 2PA-mutExon PoNe donor (abbreviated as kctd10 PoG-Ne donor) and the strategy of targeted insertion and conditional knockout using the CRISPR/Cas system. Primers K10qF and K10qR are used for qRT-PCR in L and M. (E) Images of a 10 hpf F₀ zebrafish embryo after the injection of the kctd10 PoG-Ne donor together with zCas9 mRNA and corresponding gRNAs. White arrows indicate tdGFP signals. Scale bar, 200 μm. (F) Junction PCR to detect NHEJ-mediated knockin events in the injected founder embryos. Injected: Donor+Cas9/gRNA-injected embryos. Donor: kctd10 PoG-Ne donor plasmid. Uninjected: Uninjected embryos. (G) Images of a 10 hpf F₁ zebrafish embryo from an outcross of the kctd10 PoG-Ne donor KI-positive F₀ female (#32) shown in Supplementary file 4, bearing the kctd10^PoG-Ne-1 allele. Strong maternal expression of tdGFP can be clearly observed in this F₁ embryo. Scale bar, 200 μm. (H) Schematic diagram of the kctd10 KI allele, showing the position of the primers used for junction PCR in I-K and qRT-PCR in L. A new primer pair was used to amplify the 3’ junction of the F₁ embryos. (I) Junction PCR to detect the knockin allele in individual F₁ embryos (1-4) from the cross in G. Note that not all of the embryos inherited the knockin allele from the F₀ female, indicating germline mosaicism of this adult fish. (J) Sequencing results of the PCR products from the two positive embryos (2 and 3) in I, which showed the same junction sequence of the kctd10^PoG-Ne-1 allele. (K) Sequencing results of the PCR products (using the same primer pair as in I and J) from an EGFP-positive F₁ zebrafish embryo obtained from an outcross of the positive F₀ male (#5), representing the junction sequence of the kctd10^PoG-Ne-2 allele. (L) qRT-PCR results showing the transcription level of the kctd10 locus in wild-type (WT) and kctd10 PoG-Ne donor KI zebrafish embryos at 72 hpf, using K10qF and K10qR primers. The kctd10^+/Ne-1 and kctd10^+/PoG-Ne-1 embryos were obtained from the cross of kctd10 ^{PoG-Ne-1/PoG-Ne-1} homozygotes with wild-type zebrafish with or without the injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. (M) qRT-PCR results using K10qF and K10qR primers, showing the transcription level of the kctd10 locus in the kctd10^+/Ne-1 and kctd10^Ne-1/Ne-1 embryos derived from the Cre mRNA-injected kctd10^+/PoG-Ne-1 and kctd10^{PoG-Ne-1/PoG-Ne-1} embryos, respectively. The original embryos were obtained from the crossing of kctd10^{PoG-Ne-1/PoG-Ne-1} homozygotes with kctd10^+/PoG-Ne-1 heterozygote zebrafish. The expression levels in the KI embryos were normalized to the WT ones. Data are presented as the mean ±s.d., and a two-tailed Student’s t-test was applied to calculate p values in all the experiments. *: p<0.05. **: p<0.01. ***: p<0.001. NS: Not significant.