Ge (giant egg) is a sex-linked mutation in Bombyx mori which leads to the production of large-sized eggs caused by an increase in the number of follicular epithelial cells, each with decreased cell size. We previously determined that a gene orthologous to human Phytanoyl-CoA dioxygenase domain-containing 1 (PHYHD1) is specifically disrupted in Ge and Ge 2 mutants through positional cloning of the Ge locus. However, little is known about the function of the BmPHYHD1 gene in determination of the egg size. In this study, we knocked out BmPHYHD1 using CRISPR/Cas9. We employed a novel knockout protocol developed in other lepidopteran species in which a commercialized Cas9 mixed with two sgRNAs designed for a target gene is injected into newly-laid eggs. First, we confirmed that the protocol was applicable to B. mori with high efficiency. Second, we targeted the BmPHYHD1 gene and obtained G 0 moths laid large size eggs. CRISPR/Cas9-mediated mutations were confirmed in G 1 moths that laid large-sized eggs, demonstrating that the BmPHYHD1 gene controls egg size in B. mori. Finally, the dominant/recessive relationship between Ge and + Ge is discussed and we propose the use of gi (giant egg) and gi 2 (giant egg 2) to represent giant egg mutations, Ge and Ge 2 , respectively. We believe that Ge mutants are an appropriate model for the study of genetic mechanisms mediating the size and number of cells in animals.
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