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.

The heat shock response is important for the viability of all living organisms. It involves the induction of heat shock proteins whose expression is mainly regulated by heat shock factor 1 (HSF1). Medaka ( Oryzias latipes ) is a teleost fish with an XX/XY sex determination system. High water temperature (HT) inhibits the female-type proliferation of germ cells and induces the masculinisation of XX medaka in some cases during gonadal sex differentiation. Here, we investigated the roles of HSF1 on the proliferation of germ cells using HSF1 knockout medaka. Loss of HSF1 function under HT completely inhibited the female-type proliferation of germ cells, induced the expression of the anti-Mullerian hormone receptor type 2 ( amhr2 ) and apoptosis-related genes, and suppressed that of the dead end ( dnd ) and heat shock protein-related genes. Moreover, the loss of HSF1 and AMHR2 function under HT recovered female-type proliferation in germ cells, while loss of HSF1 function under HT induced gonadal somatic cell apoptosis during early sex differentiation. These results strongly suggest that HSF1 under the HT protects the female-type proliferation of germ cells by inhibiting amhr2 expression in gonadal somatic cells. These findings provide new insights into the molecular mechanisms underlying environmental sex determination.

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