Figure 1—figure supplement 1.

Figure 1—figure supplement 1.

(A) The position and sequence of the tbx5a intron 2 (I2) target site designed for the Cas9/gRNA system. The protospacer sequence is shown in red, and the PAM is shown in green. (B) Indel efficiency evaluated by PCR and BtgI restriction endonuclease digestion. (C) Sequencing results of the uncut PCR products (corresponding to indel mutations) from B after cloning. (D) Mosaic expression of tdTomato in the heart (white arrowhead) and fin (white arrows) in a founder embryo after the injection of gRNAs purified by LiCl precipitation together with zCas9 mRNA and the tbx5a PoR-Ne donor from Figure 1A. Scale bar, 200 μm. (E) Junction PCR to detect NHEJ-mediated knockin events in founder embryos. The expected products (870 bp and 570 bp) were obtained by amplification with the corresponding primers shown in Figure 1A. Injected: Donor+Cas9/gRNA-injected embryos. Donor: tbx5a PoR-Ne donor plasmid. Uninjected: Uninjected embryos. (F) The expression of tbx5a in the zebrafish heart shown by whole-mount in situ hybridization (ventral view). The dotted lines denote the outline of the heart. Scale bar, 100 μm.