Fig. 2.

Homology and CRISPR-Cas9 targeting of zebrafish pheta1 and pheta2. (A) The domain structures of human PHETA1, human PHETA2, and the zebrafish PHETA homologs Pheta1 and Pheta2. The PH domain, coiled-coil domain, PPPxPPRR motif and OCRL binding site are highlighted. Like human PHETA2, zebrafish Pheta2 lacks the PPPxPPRR motif. (B,C) Genomic organization of human PHETA1 (B) and PHETA2 (C) and their respective homologs in mouse and zebrafish. (D) Phylogenic tree of PHETA proteins. Units indicate the number of amino acid substitutions per site. (E,F) The diagrams on the top illustrate the genomic structures of pheta1 and pheta2, with the guide RNA (gRNA) target marked with the asterisk. Green arrows indicate the primers for RT-PCR. Sequences at the bottom show the gRNA target (underlined) associated with the protospacer adjacent motif (PAM; boxed letters), and the sequences mutated by CRISPR-Cas9 (blue). (G,H) CRISPR-mediated mutations result in reading frame shifts, causing aberrant protein sequences (gray regions) and premature termination of the protein sequences for Pheta1 (G) and Pheta2 (H). The start of frameshift mutation is indicated by the red lines. (I) Normalized counts of pheta1 and pheta2 transcripts. (J,K) RT-PCR amplification of pheta1 (J) and pheta2 (K) complementary DNA (cDNA) from 4 dpf WT, pheta1^−/− and pheta2^−/− animals, using the primer pairs indicated in E and F. Two lanes from two separate pools of animals are shown for each genotype. No alternative splice forms were detected in the mutants. Expected sizes for pheta1: 326 bp (WT and pheta2^−/−) and 288 bp (pheta1^−/−). Expected sizes for pheta2: 825 bp (WT and pheta1^−/−) and 814 bp (pheta2^−/−).