Abstract: Aromatase is the key enzyme for the synthesis and conversion of estrogens from androgens.Brain type aromatase(cyp19b) is predominantly expressed and displays a sexual dimorphism in adult brain, in contrast to a slight expression with no sexual dimorphism in adult gonad of medaka (Oryzias latipes). In order to explore the bio-function ofcyp19bgene in brain and gonad development, we used CRISPR/Cas9 system to targeted knockoutcyp19b. Two targeting sites were designed at exon 2 and exon 4, respectively.Microinjection of aliquots containing gRNAs and Cas9 protein was carried out into 1-cell fertilized eggs. Results showed that two heritablecyp19bmutant alleles were successfully obtained, and twocyp19bknockout mutant homozygous lines were selected through heterozygous mating, genomic PCR and DNA sequencing analysis. There was no significant male-or female-bias in both lines ofcyp19bknockout homozygotes. In both lines ofcyp19bknockout homozygotes, the embryo could develop normally and the adult could reproduce normally, suggesting that the loss ofcyp19bgene might not directly affect the sexual differentiation of gonads.

Key words: medaka;cyp19b, gene knockout, CRISPR/Cas9