Generation of Ectodysplasin A (eda)-targeted Knockout Zebrafish Via the CRISPR/Cas9 System

Ectodysplasin A (EDA) plays a vital role in the development of skin appendages, especially in fish scales. Zebrafish model with the mutation of eda was found using CRISPR/Cas9 system. CRISPR/Cas9 nucleases targeting to two loci in exon 4 of eda, were constructed and injected into zebrafish embryos, respectively. CRISPR-Cas9 mediated mutation frequency toward eda exon 4 was approximately 16%, which was relatively low compared with that of other genes in zebrafish. Five eda mutant types were obtained in FO generation including a deletion of 5 bp, 6 bp, 8 bp and 87 bp, and an insertion of 11 bp around the targeted site respectively, and all of which happened just in one allele. But the scales of all F0 founders were normal compared with their wild counterparts. In the F1 generation, five scale loss mutants with few scales covered were achieved that were all caused by bi-allelic 11-bp insertion in eda. The insertion results in frameshift mutation of eda and leads to loss of expression and function inactivation of EDA as determined by western blotting. This provides a good model for elucidating the function of EDA in the development of fish scales.