Figure 2—figure supplement 1.

(A) The position and sequence of the tbx5a exon 3 (E3) 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 AluI restriction endonuclease digestion. The result indicates that the indel efficiency is nearly 90%. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Approximately 25% of embryos from the incross of tbx5a^+/Δ5 heterozygotes showed defects in heart (black arrows) and pectoral fins (black arrowheads). Genotyping results revealed that all the defective embryos were tbx5a^Δ5/Δ5 homozygotes (lower panel), while the siblings showed a normal morphology. The Tg(cmlc2:EGFP) transgenic background was introduced to reveal the heart morphology, and all the defective embryos also showed failure of cardiac looping. The dotted lines denote the outline of the heart. Scale bar, 200 μm. (E) qRT-PCR results showing the transcription level of the tbx5a locus in wild-type (WT) and tbx5a PoR-Ne donor KI zebrafish embryos at 72 hpf, using T5qF and T5qR primers. The tbx5a^+/Ne and tbx5a^+/PoR-Ne embryos were obtained from the crossing of the tbx5a ^{PoR-Ne/PoR-Ne} homozygotes with wild-type zebrafish with or without injection of Cre mRNA, respectively. The average expression level of wild-type embryos was set as 1. (F) qRT-PCR results using T5qF and T5qR primers, showing the transcription level of the tbx5a locus in the tbx5a^+/Ne and tbx5a^Ne/Ne embryos derived from the Cre mRNA-injected tbx5a^+/PoR-Ne and tbx5a^{PoR-Ne/PoR-Ne} embryos, respectively. The original embryos were obtained from the crossing of tbx5a^{PoR-Ne/PoR-Ne} homozygotes with tbx5a^+/PoR-Ne 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<0.001. NS: Not significant.