Fig. 2

Characterization of L-ARKO zebrafish. (A–D) Images of zebrafish larvae after whole mount in situ hybridization. 4 dpf L-ARKO larvae were subjected to whole mount in situ hybridization against the ceruloplasmin probe (A), cas9 antisense probe (B) and cas9 sense probe (C). 4 dpf WT larvae was examined against cas9 antisense probe (D). (E) PCR examination of cas9DNA in the liver, tail fin and heart of the F2 L-ARKO zebrafish. WT liver was used as a negative control. (F) T7E1 mutagenesis assay for the CRISPR target site in the ar gene in the WT zebrafish liver and F2 L-ARKO zebrafish liver, tail fin and heart. both WT and mutant ar bands are indicated with arrowheads. (G) Sequences of ar gRNA target sites. The target sites were amplified by PCR from liver DNA of an F2 L-ARKO fish and sequenced. The gRNA targeted sequence is underlined and the mutations in red. The type of mutation and frequency in percentage are indicated. All of the mutated sequences caused reading frame changes and premature termination codons.