Generation of biallelic F0 mutants in medaka using the CRISPR/Cas9 system.

Several animal models generated by genome editing methods develop somatic mosaic mutations including wild-type genome sequence in F0 generation because it is difficult to use editing tools at the one-cell stage. Producing complete knockout animals quickly is a great advantage in determining the function of target genes. This study investigated the generation of F0 knockout medaka using the CRISPR/Cas9 system. To determine whether this editing system induced mutations in the medaka genome at the one-cell stage, recombinant Cas9 protein, tracrRNA and crRNA for dead end (dnd), which is essential for germ cell development, were injected into one-cell stage embryos of olvas-DsRedExpress transgenic medaka. This allowed germ cells to be visualized by DsRed fluorescence. Genomic DNA extracted from embryos at the one-cell stage was analyzed by sequencing. Predictably, biallelic mutated sequence patterns in the target sites of dnd were found in the injected embryos. To investigate the phenotypes of the mutated fish, fluorescent and histological observations of germ cells were carried out using fry and adults. The mutations resulted in a complete loss of germ cells, suggesting loss of function of dnd in the injected embryos. Therefore, this system appears to be extremely effective for the production of F0 knockout medaka.